Alpha 1,2 fucosyltransferase

ABSTRACT

A bacterial α1,2-fucosyltransferase gene and deduced amino acid sequence is provided. The gene is useful for preparing α1,2-fucosyltransferase polypeptide, and active fragment thereof, which can be used in the production of oligosaccharides such as Lewis X, Lewis Y, Lewis B and H type 1, which are structurally similar to certain tumor-associated carbohydrate antigens found in mammals. These product glycoconjugates also have research and diagnostic utility in the development of assays to detect mammalian tumors.

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field ofα1,2-fucosyltransferases and, more specifically, toα1,2-fucosyltransferase polypeptides.

BACKGROUND OF THE INVENTION

[0002]Helicobacter pylori is an important human pathogen which causesboth gastric and duodenal ulcers and has also been associated withgastric cancer and lymphoma. This microorganism has been shown toexpress cell surface glycoconjugates including Lewis X, Lewis Y, andsialyl Lewis X. These bacterial oligosaccharides are structurallysimilar to tumor-associated carbohydrate antigens found in mammals.

[0003] The presence of H. pylori isolate has been associated with anincreased risk for development of gastric cancer (Wirth, H.-P., Yang,M., Karita, M., and Blaser, M. J. (1996) Infect. Immun. 64, 4598-4605).This pathogen is highly adapted to colonize human gastric mucosa and mayremain in the stomach with or without causing symptoms for many years.Although H. pylori elicits local as well as systemic antibody responses,it escapes elimination by the host immune response due to itssequestered habitation within human gastric mucosa. Another mechanism bywhich H. pylori may protect itself from the action of the host immuneresponse is the production of surface antigens mimicking those in thehost.

[0004] In mammalian cells the enzyme fucosyltransferase (namely FucT)catalyzes the last step in the synthesis of two carbohydrate structures,Galβ 1-4[Fucα1-3] GlcNAc (Lewis X, Le^(x) for short) or NeuAcα2-3-Galβ1-4[Fucα1-3]GlcNAc (sialyl Lewis X, sLe^(x) for short). (Lowe et al.,1990, Cell 57: 475-484.; Kukowska-Latallo et al., 1990, Genes &Development 4:1288-1303.) Cell surface α(1,3)- and α(1,2)-fucosylatedoligosaccharides, that is, Lewis X (Le^(x)), sialyl Lewis X (sLe^(x))and Lewis Y (Le^(y)), are present on both eukaryotic and microbial cellsurfaces. In mammals, Le^(x) is a stage-specific embryonic antigen,however, Le^(x), sLe^(x) and Le^(y) are also regarded astumor-associated markers. The biological functions of these bacterialoligosaccharide structures are not fully understood. It has beensuggested that such glycoconjugates produced by H. pylori, may mimichost cell antigens and could mask the bacterium from the host immuneresponse. It is also possible that these bacterial Lewis antigens coulddown regulate the host T-cell response. Therefore, production of suchantigens may contribute to colonization and long-term infection of thestomach by H. pylori.

[0005] Presently, use of carbohydrates as potential therapeutic drugshas become popular in the field of medical chemistry. In addition,qualitative and quantitative carbohydrates including Le^(x), Le^(y) andsLe^(x) are also required as reagents for assaying the enzymes which areinvolved in the biosynthesis of glycoconjugates in cells. Le^(x), Le^(y)and sLe^(x) products which are commercially available are chemicallysynthesized. However, synthesis of these products gives rise to severallimitations such as time-consuming, complicated procedures and lowyields. Although several mammalian fucosyltransferases have been clonedand expressed, enzymatic synthesis of Le^(x), Le^(y) and sLe^(x)products for a commercial purpose has not been reported.

[0006] The whole genome sequence of H. pylori 26695 had been published,which will undoubtedly facilitate the genetic studies of H. pylori. H.pylori genome sequence revealed the existence of two copies ofα(1,3)fucT gene, whereas no putative α(1,2) fucT gene had beenannotated.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the discovery of aα1,2-fucosyltransferase polypeptide and gene which encodes thepolypeptide. The gene was expressed in vitro and a mutagenesis studydemonstrated that this gene is involved in Le^(Y) synthesis. The presentinvention includes a polynucleotide sequence encodingα1,2-fucosyltransferase polypeptide which is useful in the detection andsynthesis of α1,2-fucosyltransferase polypeptide, and anα1,2-fucosyltransferase that is able to synthesize Le^(Y), Le^(B) and Htype 1 structures.

[0008]Helicobacter pylori lipopolysaccharide (LPS) express humanoncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Yinvolves the actions of α(1,3) and α(1,2)fucosyltransferases (FucTs).Disclosed herein are the molecular cloning and characterization of genesencoding H. pylori α(1,2)FucT (Hp fucT2) from various H. pylori strains.Also provided are constructed Hp fucT2 knock-out mutants thatdemonstrate the loss of Lewis Y production in these mutants by ELISA andimmunoelectron microscopy. The α1,2 fucT2 gene contains a hypermutablesequence (poly C and TAA repeats) which provides a possibility offrequent shifting into and out of coding frame by a polymerase slippagemechanism. Thus, α1,2 fucT2 gene displays two major genotypes: eitherencoding a single full-length open reading frame (ORF, as in the strainUA802), or truncated ORFs (as in the strain 26695). In vitro expressionof Hp fucT2 genes demonstrated that both types of the gene have apotential to produce the full-length protein. The production of thefull-length protein by the 26695 fucT2 gene could be attributed totranslational—1 frameshifting, since a perfect translation frameshiftcassette resembling that of Escherichia coli dnaX gene is present. Theexamination of the strain UA1174 revealed that its fucT2 gene has aframeshifted ORF at the DNA level which cannot be compensated bytranslation frameshifting, accounting for its Lewis Y -off phenotype. Inanother strain, UA1218, the fucT2 gene is turned off apparently due tothe loss of its promoter. Based on these data, we proposed a model forthe variable expression of Lewis Y by H. pylori, in which the regulationat the level of replication, transcription, and translation of the fucT2gene may all be involved.

[0009] In another embodiment, the invention provides a method of usingthe novel α1,2-fucosyltransferase to synthesize oligosaccharides such asLe^(x), Le^(y), sLe^(x), Le^(A), Le^(B), H type 1 and H type 2.

[0010] In another embodiment the invention provides the novelpolypeptide of α1,2-fucosyltransferase which is useful in thedevelopment of antibodies to α1,2-fucosyltransferase.

[0011] In another embodiment, a polypeptide of α1,2-fucosyltransferasehaving a frameshift variant resulting from a “slippery” heptanucleicacid sequence X XXY YYZ, wherein X=C or A, Y=T or A and Z=A or G (e.g.,A AAA AAG) is provided. In another embodiment, theα1,2-fucosyltransferase is a polypeptide which has a sequence of SEQ IDNO:2. In another embodiment the polynucleotide sequence encodingα1,2-fucosyltransferase has a variable number of poly-cytosine repeatsand TAA repeats in different H. pylori strains.

[0012] Further provided is a method for producingα1,2-fucosyltransferase. The method involves the step of culturing agene expression system which comprises a host cell which has beenrecombinantly modified with a polynucleotide encodingα1,2-fucosyltransferase or a portion thereof and harvesting theα1,2-fucosyltransferase. A preferred embodiment of the method isdirected to the use of the claimed genetic expression system whichproduces α1,2-fucosyltransferase.

[0013] Further provided is a method to measure the enzymatic activityand acceptor specificity of α1,2-fucosyltransferase. The method involvesthe use of a structurally defined oligosaccharide substrate (acceptor)in a radioactive labeled assay system and identification of the reactionproducts by capillary electrophoresis. In another embodiment, anα1,2-fucosyltransferase has a substrate specificity that is distinctfrom the conventional α1,2-fucosyltransferase of mammalian origin anduses a different pathway to synthesize Lewis antigens.

[0014] Also provided are knockout organisms in which expression ofα1,2-fucosyltransferase has been prevented or in which theα1,2-fucosyltransferase expression results in a polypeptide lacking wildtype biological activity.

[0015] These and many other features and attendant advantages of thepresent invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the Examples.

ABBREVIATIONS

[0016] The abbreviation used are: α1,2-FucT, α1,2-fucosyltransferaseunless specified otherwise; Le^(x), Lewis X; sLe^(x), sialyl-Lewis X;Le^(y), Lewis Y; Le^(B), Lewis B; nt, nucleotide (s); kb, kilobase (s);aa, amino acid (s); PCR, polymerase chain reaction; ORF, open readingframe; RSB, a ribosomal binding site; LPS, lipopolysaccharides;LacNAc-R, Galβ1-4GlcNAcβ-O—(CH₂)₈COOMe;Galβ1-3GlcNAc-R,Galβ1-3GlcNAcb-O—(CH²)⁸COOMe; LacNAc-TMR,Galβ1-4GlcNAcβ-O—(CH₂)₈CO—NHCH₂CH₂NH—TMR.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the nucleotide sequence analysis of Hp fucT2. (A)Gene organization of Hp fucT2 region in the genome of H. pylori 26695and UA802. GW44 and GW32 indicate the two primers used for cloning HpfucT2 genes. (B) Nucleotide sequences of the center region of Hp fucT2showing the features (simple repeats) responsible for frameshift betweenprototype (UA802) and variant type (26695) genes. The divergentnucleotides between the two sequences are marked by “x”. Due to thedifferent repeat number of poly C residues, the initiating reading frameof 26695 fucT2 (HP0094) encounters a TGA stop codon (marked withasterisks) shortly after the poly-C region. About 110 bp furtherdownstream, there appears a potential start codon ATG (marked with dots)in the −1 frame (HP0093), which is the same as the reading flame of 802fucT2. The three putative X XXY YYZ motifs (X, Y, and Z representspecific nucleotides in a particular reading from) are given in boldface and underlined. Additional elements for programmed translationframeshift in 26695 fucT2 resembling those in E. coli dnaX gene are alsounderlined. (C) The putative 26695 fucT2 translation frameshiftcassette. Shown is the mRNA structure deduced from the DNA sequence inline 2 of (B). The AAAAAAG heptamer (bold) is a highly slippery sequenceidentified in other DNA sequences. UGA (sidelined in the stem structure)is the stop codon in the initiating frame (0 frame). SD indicates aninternal Shine-Dalgarno-like sequence. According to the E. coli dnaXframeshift model, AAAAAAG sequence is the frameshift site, and bothupstream SD sequence and downstream stem-loop structure enhanceframeshifting. (D) Shows the amino acid sequence and nucleic acidsequence for α1,2 fucosyltransferase.

[0018]FIG. 2 shows an analysis of the deduced Amino acid (aa) sequenceof Hp fucT2. (A) Schematic representation of the domain structures ofmammalian and bacterial α(1,2) fucosyltransferases. Cyt, cytoplasmic.TM, transmembrane. Hatched boxes represent three highly conserved aasequence motifs. (B) Alignment of the three motifs of aa sequences whichare highly conserved in all prokaryotic and eukaryoticα(1,2)fucosyltransferases. The length (in aa) of each protein is givenin parentheses after the name of organisms, and the positions of eachmotif within the protein are labeled in parentheses after each aminoacid sequence. Ye, Y. enterocolitica. Ll, Lactococcus lactis. Accessionnumbers of these sequences are: M35531 (man FUT1), U17894 (man FUT2),AF076779 (Hp FucT2, from the prototype fucT2 of UA802), U46859 (YeWbsH), and U93364 (Ll EpsH).

[0019]FIG. 3 shows the cloning and in vitro expression of Hp fucT2genes. (A) Plasmid constructs containing intact or partial Hp fucT2gene. Heavy arrows represent the predicted ORFs, and the thin linesindicate the flanking regions that had been cloned together with thecoding region into the vector. The small arrows point to the directionof the transcription from the T7 promoter. Restriction endonucleasesites HindIII (H) and EcoRI (E) were used for constructing CAT insertionmutants. (B) Autoradiograph of a 0.1% SDS-12% PAGE analyzing the proteinsynthesis products from various plasmid constructs by E. coli T7 S30extract. Lane 1, no DNA template. Some protein bands are fromtranscription-translation of endogenous DNA or RNA in the cell extract.Lane 2, pGEM-T vector. Lane 3, 4, and 5, plasmid constructs pGEMB3,pGEMH2, and pGEMI6, respectively. The full length protein (33 KD) markedby the large arrow was overexpressed from intact fucT2 genes but notfrom 5′-truncated gene. A half-length protein (17 KD, marked by thesmall arrow) was also produced from 26695 fucT2, but not from 802 fucT2.Lane 6, 7, and 8, pGHC26, pGEC26, and pGHC8, plasmid mutants with CATinsertion at HindIII site of 26695 fucT2, at EcoRI site of 26695 fucT2.and at HindIII site of UA802 fucT2, respectively. All three plasmidmutants gave rise to strong expression of 24 KD CAT protein. Themolecular mass markers (Life Technologies, Inc) are indicated on theright.

[0020]FIG. 4 shows a transmission electron micrographs of H. pyloriUA802 and its isogenic mutant carrying CAT insertion within the fucT2gene at HindIII (ΔH). Cells were incubated with anti-Le^(Y) MAb and goatanti-mouse IgM-10 nm colloidal gold particles. Gold particles werepresent on the wild type cell (both on the cell wall and flagellasheath, marked by arrowheads) but absent on the mutant cell.

[0021]FIG. 5 shows two possible pathways for the synthesis of Lewis Y inH. pylori.

[0022]FIG. 6 shows an immunoblots of H. pylori LPS for detection ofLewis structures. Proteinase K treated whole cells extracts of H. pylori26695 and UA802 wild type strains (WT) and their isogenic mutants (ΔHand ΔE) were resolved on SDS-PAGE and electroblotted onto anitrocellulose membrane, and the LPS were immunostained usinganti-Le^(Y) (A) or anti-Le^(X) antibody (B).

[0023]FIG. 7 shows identification of the reaction products of Hpα1,2-fucosyltransferase by capillary electrophoresis. The enzyme usedhere was the overexpressed UA802 α1,2-fucosyltransferase polypeptide.The reactions were carried out as described in Example 3 below. (A) Thereaction of type 2 substrates LacNac (line a) and Le^(X) (line b). (B)The reactions on Type 1 substrates (line d) and Le^(B) (line e). Line cand f represent the standard TMR-labeled oligosaccharides: (1) linkingarm, (2) GlcNAc, (3) LacNAc, (4) H type 2, (5) Le^(X), (6) Le^(Y), (7)Type 1, (8) H type 1, (9) Le^(A), and Le^(B). All electropherograms areY-offset for clarity.

[0024]FIG. 8 shows identified pathways for the synthesis of Lewisantigens in H. pylori. Lewis structures known to be expressed on the H.pylori cell surface are boxed. Solid arrows represent thefucosyltransferase activities that have been demonstrated in this study,and the thickness of the arrows indicates the relative level of theenzyme activity. (A) H. pylori strains predominantly express Le^(X) andLe^(Y), and do not appear to express H type 2. It seems reasonable thatH. pylori utilizes Le^(X) to synthesize Le^(Y). For operation of thispathway H. pylori normally maintains a higher level ofα1,3-fucosyltransferase than of α1,2-fucosyltransferase. (B) H. pyloriα1,2-fucosyltransferase has the ability to transfer fucose to Type 1 aswell as to Le^(A). The synthesis of Le^(B) requires the concerted actionof α1,2-fucosyltransferase with an α1,4-fucosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a purifiedα1,2-fucosyltransferase polypeptide, polynucleotide which encode theα1,2-fucosyltransferase, and the use of the α1,2-fucosyltransferase geneand α1,2-fucosyltransferase polypeptide in the production of biologicsand in the screening of biological tissues and fluids. The inventionalso relates to antibodies against α1,2-fucosyltransferase polypeptidesand their use in diagnosing disorders and in monitoring disease.

[0026] The α1,2-fucosyltransferase Polypeptide

[0027] The amino acid sequence encoded by the α1,2-fucosyltransferasegene is shown in FIG. 1D (SEQ ID NO:2). Because theα1,2-fucosyltransferase are prokaryotically derived post-translationalmodifications are not made to the enzyme, unlike the eukaryoticallyexpressed α1,2-fucosyltransferase.

[0028] Additionally, the α1,2-fucosyltransferase polypeptide may bealtered by addition, substitution or deletions of peptide sequences inorder to modify its activity. For example, polypeptide sequences may befused to the α1,2-fucosyltransferase polypeptide in order to effectuateadditional enzymatic activity. Alternatively, amino acids may be deletedor substituted to remove or modify the activity of the protein. Theprotein may be modified to lack α1,2-fucosyltransferase enzymaticactivity, but retain its three-dimensional structure. Such modificationwould be useful in the development of antibodies againstα1,2-fucosyltransferase polypeptide as described more fully below.

[0029] In yet another embodiment, the invention includes aspects of theenzymatic activity of α1,2-fucosyltransferase, wherein theα1,2-fucosyltransferase polypeptide lacks α1,4-fucosyltransferase orα1,3-fucosyltransferase activity or lacks both α1,3-fucosyltransferaseand α1,4-fucosyltransferase activity.

[0030] The α1,2-fucosyltransferase gene product may include thosepolypeptides encoded by the α1,2-fucosyltransferase gene sequencesdescribed in the section below. Specifically, α1,2-fucosyltransferasegene products, sometimes referred to herein as “α1,2-fucosyltransferasepolypeptide”, may include α1,2-fucosyltransferase gene product encodedby an α1,2-fucosyltransferase gene sequence shown in FIG. 1 and SEQ IDNO:1, as well as different versions of the gene sequences deposited inGenBank under the accession numbers AF093828-AF093833. Thus, the term“α1,2-fucosyltransferase polypeptide” includes full length expression aswell as polypeptides, such as smaller peptides, which retain abiological activity of the full length product, such asα1,2-fucosyltransferase activity.

[0031] In addition, α1,2-fucosyltransferase gene products may includeproteins or polypeptides that represent functionally equivalent geneproducts. Such an equivalent α1,2-fucosyltransferase gene product maycontain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the α1,2-fucosyltransferasegene sequences described above, but which results in a silent change,thus producing a functionally equivalent α1,2-fucosyltransferase geneproduct. Amino acid substitutions may be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved.

[0032] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; planar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa polypeptide capable of exhibiting a substantially similar in vivoactivity as the endogenous α1,2-fucosyltransferase gene products encodedby the α1,2-fucosyltransferase gene sequences described above, as judgedby any of a number of criteria, including but not limited toantigenicity, i.e., the ability to bind to ananti-α1,2-fucosyltransferase antibody, immunogenicity, i.e., the abilityto generate an antibody which is capable of binding aα1,2-fucosyltransferase protein or polypeptide, as well as enzymaticactivity. For example, the frameshift mutant resulting from expressionof the sequence XXXYYYZ results in a product which may retain antigenicproperties similar to those of wild type α1,2-fucosyltransferase.

[0033] A substantially purified α1,2-fucosyltransferase protein,polypeptide, and derivative (including a fragment) is substantially freeof other proteins, lipids, carbohydrates, nucleic acids, and otherbiological materials with which it is naturally associated. For example,a substantially purified functional fragment of α1,2-fucosyltransferasepolypeptide can be at least 60%, by dry weight, the molecule ofinterest. One skilled in the art can purify a functional fragment ofα1,2-fucosyltransferase protein using standard protein purificationmethods and the purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography), and amino-terminal amino acid sequence analysis.

[0034] Included within the scope of the invention areα1,2-fucosyltransferase proteins, polypeptides, and derivatives(including fragments) which are differentially modified during or aftertranslation. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc. Additionally,the composition of the invention may be conjugated to other molecules toincrease their water-solubility (e.g., polyethylene glycol), half-life,or ability to bind targeted tissue.

[0035] Furthermore, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into theα1,2-fucosyltransferase polypeptide sequence. Non-classical amino acidsinclude, but are not limited to, the D-isomer of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,γ-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids, such as β-methyl amino acids,α-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

[0036] While random mutations can be made to α1,2-fucosyltransferase DNA(using random mutagenesis techniques known to those skilled in the art)and the resulting mutant α1,2-fucosyltransferase polypeptides tested foractivity, site-directed mutation of the α1,2-fucosyltransferase codingsequence can be engineered (using site-directed mutagenesis techniqueswell known to those skilled in the art) to create mutantα1,2-fucosyltransferase polypeptides with increased functionalcharacteristics.

[0037] Polypeptides corresponding to one or more domains of theα1,2-fucosyltransferase protein, truncated or deletedα1,2-fucosyltransferase proteins, as well as fusion proteins in whichthe full length α1,2-fucosyltransferase proteins, polypeptides, orderivatives (including fragments), or truncated α1,2-fucosyltransferase,is fused to an unrelated protein, are also within the scope of theinvention and can be designed on the basis of theα1,2-fucosyltransferase nucleotide and α1,2-fucosyltransferase aminoacid sequences disclosed in this section and the section above. Thefusion protein may also be engineered to contain a cleavage site locatedbetween a α1,2-fucosyltransferase sequence and thenon-α1,2-fucosyltransferase protein sequence, so that theα1,2-fucosyltransferase polypeptide may be cleaved away from thenon-α1,2-fucosyltransferase moiety. Such fusion proteins or polypeptidesinclude but are not limited to IgFc fusion which may stabilize theα1,2-fucosyltransferase protein in vivo; or fusion to an enzyme,fluorescent protein, or luminescent protein which provide a markerfunction.

[0038] The α1,2-fucosyltransferase polypeptide may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the α1,2-fucosyltransferase polypeptides of theinvention by expressing a nucleic acid containingα1,2-fucosyltransferase gene sequences are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing α1,2-facosyltransferase codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,Molecular Cloning, a Laboratory Manual, Cold Springs Harbor Press, N.Y.,and Ausubel F. M. et al., eds., 1989, Current Protocols in MolecularBiology, Vol. 1, Green Publishing Associates, Inc., and John Willey &Sons, Inc., New York. Alternatively, RNA capable of encodingα1,2-fucosyltransferase polypeptide may be chemically synthesized using,for example, synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety. The use ofsuch synthetic peptide fragments of α1,2-fucosyltransferase forgenerating polyclonal antibodies is described below.

[0039] The α1,2-fucosyltransferase Gene

[0040] The α1,2-fucosyltransferase gene (FIG. 1) is expressed in H.pylori. Nucleic acid sequences of the identified α1,2-fucosyltransferasegenes are described herein. As used herein, “α1,2-fucosyltransferasegene” refers to (a) a gene containing the DNA sequence shown in FIG. 1;(b) any DNA sequence that encodes the amino acid sequence shown in FIG.1D, SEQ ID NO: 2; (c) any DNA sequence that hybridizes to the complementof the coding sequences shown in FIG. 1, SEQ ID NO: 1, under stringentconditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989, CurrentProtocols in Molecular Biology, Vol. 1, Green Publishing Associates,Inc., and John Willey & Sons, Inc., New York, at p. 2.10.3) and encodesa gene product functionally equivalent to a gene product encoded bysequences shown in FIG. 1; and/or (d) any DNA sequence that hybridizesto the complement of the coding sequences disclosed herein (as shown inFIG. 1), under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2% SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), and encodes a gene product functionally equivalent toa gene product encoded by sequences shown in FIG. 1.

[0041] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the DNA sequences (a) through (c), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act at α1,2-fucosyltransferase generegulation and/or as antisense primers in amplification reactions ofα1,2-fucosyltransferase gene nucleic acid sequences. Further, suchsequences may be used as part of ribozyme and/or triple helix sequences,also useful for α1,2-fucosyltransferase gene regulation. Still further,such molecules may be used as components of diagnostic methods wherebythe presence of a pathogen or metastatic tumor cell may be detected.

[0042] The invention also encompasses (a) DNA vectors that contain anyof the foregoing coding sequences and/or their complements (e.g.,antisense); (b) DNA expression vectors that contain any of the foregoingcoding sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

[0043] The invention includes fragments of any of the DNA sequencesdisclosed herein. Fragments of the α1,2-fucosyltransferase genecorresponding to coding regions of particular domains, or in which oneor more of the coding regions of the domains is deleted, are useful.Such α1,2-fucosyltransferase gene fragments may encode truncated geneproducts that retain a biological activity of the full-lengthα1,2-fucosyltransferase polypeptide, such as α1,2-fucosyltransferaseactivity or immunogenicity. The invention also includes mutantα1,2-fucosyltransferase genes encoding substitutions of amino acids asdescribed below.

[0044] In addition to the gene sequences described above, homologs ofsuch sequences, as may, for example, be present in other species,including humans, may be identified and may be readily isolated, withoutundue experimentation, by molecular biological techniques well known inthe art. Further, there may exist genes at other genetic loci within thegenome that encode proteins which have extensive homology to one or moredomains of such gene products. These genes may also be identified viasimilar techniques.

[0045] The α1,2-fucosyltransferase gene and its homologs can be obtainedfrom other organisms thought to contain α1,2-fucosyltransferaseactivity. For obtaining cDNA, tissues and cells in whichα1,2-fucosyltransferase is expressed are optimal. Tissues which canprovide a source of genetic material for α1,2-fucosyltransferase and itshomologs, therefore, include intestinal mucosal cells and tumorigeniccells. For example, the isolated α1,2-fucosyltransferase gene sequencesmay be labeled and used to screen a cDNA library constructed from mRNAobtained from the organism of interest. The hybridization conditionsused should be of a lower stringency when the cDNA library is derivedfrom an organism different from the type of organism from which thelabeled sequence was derived. Alternatively, the labeled fragment may beused to screen a genomic library derived from the organism of interest,again, using appropriately stringent condition. Low stringencyconditions are well known in the art, and will vary predictablydepending on the specific organism from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, a LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y.

[0046] Further, a previously unknown α1,2-fucosyltransferase gene typesequence may be isolated by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequence within the gene of interest. The template for the reaction maybe cDNA obtained by reverse transcription of mRNA prepared from human ornon-human cell lines or tissue known or suspected to express aα1,2-fucosyltransferase gene.

[0047] The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a α1,2-fucosyltransferasegene-like nucleic acids sequences. The PCR fragment may then be used toisolate a full length cDNA clone by a variety of methods. For example,the amplified fragment may be labeled and used to screen a bacteriophagecDNA library. Alternatively, the labeled fragment may be used to screena genomic library.

[0048] PCR technology may also be utilized to isolate DNA sequences,including full length cDNA sequences. For example, RNA may be isolated,following standard procedures, from an appropriate cellular or tissuesource. A reverse transcription reaction may be performed on the RNAusing an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanidines using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies which may beused, see e.g., Sambrook et al, 1989, Molecular Cloning, a LaboratoryManual, Cold Springs Harbor Press, N.Y.

[0049] In cases where the α1,2-fucosyltransferase gene identified is thenormal, or wild type, gene, this gene may be used to isolate mutantalleles of the gene. Mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes tointestinal mucosal disease and/or tumorigenicity. Mutant alleles andmutant allele products may then be utilized in the therapeutic anddiagnostic systems described below.

[0050] A cDNA of the mutant gene may be isolated, for example by PCR. Inthis case, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically the 5′ end of the normal gene. Using theseprimers, the product is then amplified via PCR, cloned into a suitablevector, and subjected to DNA sequences analysis through methods known inthe art. By comparing the DNA sequence of the mutant gene to that of thenormal gene, the mutation(s) responsible for the loss or alteration offunction of the mutant gene product can be ascertained.

[0051] A variety of host-expression vector systems may be utilized toexpress the α1,2-fucosyltransferase gene coding sequences of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit theα1,2-fucosyltransferase gene product of the invention in situ. Thesehosts include, but are not limited to, microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containingα1,2-fucosyltransferase gene product coding sequences; yeast (e.g.Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the α1,2-fucosyltransferase gene product codingsequences; insect cell systems infected with recombinant virusexpression vectors (erg., baculovirus) containing theα1,2-fucosyltransferase gene product coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing α1,2-fucosyltransferase gene product codingsequences; or mammalian cell systems (e.g., COS, SHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0052] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for theα1,2-fucosyltransferase gene product being expressed. For example, whena large quantity of such a protein is to be produced, for the generationof pharmaceutical compositions of α1,2-fucosyltransferase polypeptide orfor raising antibodies to α1,2-fucosyltransferase polypeptide, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which theα1,2-fucosyltransferase gene product coding sequence may be ligatedindividually into the vector in flame with the lac z coding region thata fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109); and the like. pGEX vectors may also beused to express foreign polypeptide as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0053] In an insect system, Autographa colifornica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperday cells. The α1,2-fucosyltransferase genecoding sequence may be cloned individually into non-essential regions(for example the polyhedrin gene) of the virus and placed under thecontrol of an AcNPV promoter. Successful insertion ofα1,2-fucosyltransferase gene coding sequence will result in inactivationof the polyhedrin gene and production of non-occluded recombinant virus.These recombinant viruses are then used to infect S. frugiperda cells inwhich the inserted gene is expressed.

[0054] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the α1,2-fucosyltransferase gene coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing α1,2-fucosyltransferasegene product in infected hosts (See Logan & Shenk, 1984, Proc. Natl.Acad. Sci, USA 81:3655-3659). Specific initiation signals may also berequired for efficient translation of inserted α1,2-fucosyltransferasegene product coding sequences. These signals include the ATG initiationcodon and adjacent sequences. In cases where an entireα1,2-fucosyltransferase gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translation control signals may be needed. However, incases where only a portion of the α1,2-fucosyltransferase gene codingsequences is inserted, exogenous translational control signals,including, the ATG initiation codon must be provided.

[0055] Transfection via retroviral vectors, naked DNA methods andmechanical methods including micro injection and electroporation may beused to provide either stably transfected host cells (i.e., host cellsthat do not lose the exogenous DNA over time) or transient transfectedhost cells (i.e., host cells that lose the exogenous DNA during cellreplication and growth).

[0056] An alternative fusion protein system allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cell infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0057] The α1,2-fucosyltransferase gene products can also be expressedin transgenic animals. Animals of any species, including, but notlimited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats,and non-human primates may be used to generate α1,2-fucosyltransferasetransgenic animals.

[0058] Expression Systems for α1,2-Fucosyltransferase

[0059] The novel bacterial α1,2-fucosyltransferase encoded by thedisclosed gene, and enzymatically active fragment thereof, can be usedin the production of fucosylated oligosaccharides such as Lewis Y(Le^(y)) and Lewis B (Le^(B)). These bacterial oligosaccharides arestructurally similar to certain tumor-associated carbohydrate antigensfound in mammals. These product glycoconjugates also have research anddiagnostic utility in the development of assays to detect mammaliantumors.

[0060] The fucosylated oligosaccharides may be produced by any number ofmethods utilizing the methods and compositions described herein.Standard enzymology techniques well known in the art may be utilized todevelop systems to provide fucosylated oligosaccharides (see for examplethe Methods in Enzymology, volume series published by Academic Press;and Tim Bugg, “An Introduction to Enzyme and Coenzyme Chemistry”, 1997,Blackwell Sciences, Inc.).

[0061] “Substrate”, as used herein, means any material or combinationsof different materials, that may be acted upon by the polypeptide of theinvention to give rise to fucosylated oligosaccharides, for example, andnot by way of limitation, substrates may include LacNAc-R andGDP-fucose.

[0062] Cells containing and cell-free systems may be used to produce thefucosylated oligosaccharides of the present invention. Cells containingand cell-free systems will be better understood in the description andexamples that follow. Such systems are useful in the development offucosylated oligosaccharides.

[0063] The present invention provides a method for synthesizingfucosylated oligosaccharides by reacting substrates in the presence ofα1,2-fucosyltransferase, capable of catalyzing the formation of thefucosylated oligosaccharides from the substrates.

[0064] The α1,2-fucosyltransferase may be used regardless of its originso long as it is capable of producing the fucosylated oligosaccharidesfrom the substrates. The source of the α1,2-fucosyltransferase may bederived according to the methods and compositions as described herein,for example, through protein purification from host cells transfectedwith an expression system as described more fully below.

[0065] The substrates are allowed to react with theα1,2-fucosyltransferase polypeptide under suitable conditions to allowformation of the enzymatic product. Suitable conditions can be easilydetermined by one skilled in the art. For example, suitable conditionswill include contacting the substrate and polypeptide for a sufficienttime and under sufficient conditions to allow formation of the enzymaticproduct, e.g. Le^(y), Le^(B). These conditions will vary depending uponthe amounts and purity of the substrate and enzyme, whether the systemis a cell-free or cellular based system. These variables will be easilyadjusted by those skilled in the art. For example, the period ofexposure of the enzyme to the substrate will be longer at lowertemperatures, e.g., 4° C. rather than at higher temperatures. In themethods for synthesizing the fucosylated oligosaccharides there are norestriction in terms of the timing of the addition of the substrates.The ratios of the various substrates should be in equal proportions,i.e. 1:1. The ratios of the enzyme to the substrates may be varieddepending upon the rate and quantity of fucosylated oligosaccharidesdesired.

[0066] The method of producing the fucosylated oligosaccharides may becarried out at temperatures of 4° C. to 60° C. Additionally, a number ofbuffers may be used, for example, and not by way of limitation, a bufferhaving a pH between 6.5 and 8.0, and in the presence of 15-30 mM Mn²⁺.After a desired amount of fucosylated oligosaccharides are produced theα1,2-fucosyltransferase polypeptide may be inactivated by heating,centrifugal separation, or the like. The resulting fucosylatedoligosaccharides may be further purified by techniques-known to thoseskilled in the art.

[0067] Cell containing systems for the synthesis of fucosylatedoligosaccharides may include recombinantly modified host cells accordingto the methods described below or may be naturally occurring cells whichexpress α1,2-fucosyltransferase polypeptide or an enzymatically activeportion thereof, so long as the cell is capable of catalyzing thesynthesis of fucosylated oligosaccharides from substrates.

[0068] In the case of cell containing systems the host cell is contactedwith the substrate, under conditions and for sufficient time to producethe oligosaccharide. The time and conditions will vary depending uponthe host cell type and culture conditions and can be easily determinedby those of skill in the art.

[0069] The invention provides a gene expression system for producingα1,2-fucosyltransferase polypeptides. The gene expression systemcomprises a host cell which has been modified with a polynucleotideencoding α1,2-fucosyltransferase polypeptide or a portion thereof, asdescribed above.

[0070] A preferred gene expression system of the invention involves hostcell modified with a polynucleotide encoding α1,2-fucosyltransferasepolypeptide or a portion thereof.

[0071] The method involves culturing a gene expression system createdaccording to the methods described above under conditions sufficient toproduce the α1,2-fucosyltransferase polypeptide. The gene expressionsystem comprises a host cell which has been recombinantly modified witha polynucleotide encoding a α1,2-fucosyltransferase polypeptide or aportion thereof.

[0072] The method is also directed to harvesting theα1,2-fucosyltransferase polypeptide. A further step of the methodinvolves substantially purifying the harvested α1,2-fucosyltransferase.The purified α1,2-fucosyltransferase polypeptide may be used in thesynthesis of fucosylated oligosaccharides or the preparation ofantibodies as described above.

[0073] Specifically disclosed herein is a gene expression systemrecombinantly modified with a DNA sequence containing theα1,2-fucosyltransferase gene. The sequence contains an open readingframe (ORF) of approximately 900 base pairs which are transcribed intoα1,2-fucosyltransferase product having a calculated molecular weight of35,193 daltons.

[0074] As used herein, the term “recombinantly modified” meansintroducing a polynucleotide encoding α1,2-fucosyltransferasepolypeptide into a living cell or gene expression system. Usually, thepolynucleotide is present in a plasmid or other vector, althoughmodification can also occur by uptake of free α1,2-fucosyltransferasepolynucleotide or numerous other techniques known in the art.

[0075] As used herein, the term “gene expression system” means a livingeukaryotic or prokaryotic cell into which a gene, whose product is to beexpressed, has been introduced, as described above.

[0076] As used herein, the term “harvesting” means collecting orseparating from the gene expression system the product produced by theinserted polynucleotide.

[0077] Polynucleotide sequences encoding α1,2-fucosyltransferasepolypeptides can be expressed by polynucleotide transfer into a suitablehost cell.

[0078] “Host cells” are cells in which a vector can be propagated andits DNA expressed. A gene expression system is comprised of a host cellin which a vector was propagated and the vector's DNA expressed. Theterm “host cell” also includes any progeny of the subject host cell. Itis understood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. However,such progeny are included when the term “host cell” is used. Host cellswhich are useful in the claimed gene expression system and the claimedmethod of producing α1,2-fucosyltransferase polypeptide includebacterial cells, yeast cells fungal cells, plant cells and animal cells.

[0079] Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art. In thepresent invention, the α1,2-fucosyltransferase polynucleotide sequencesmay be inserted into a recombinant expression vector. The term“recombinant expression vector” refers to a plasmid, virus or othervehicle known in the art that has been manipulated by insertion orincorporation of the α1,2-fucosyltransferase genetic sequences. Suchexpression vectors contain a promoter sequence which facilitates theefficient transcription of the inserted genetic sequence of the host.The expression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart. Such vectors are used to incorporate DNA sequences of theinvention.

[0080] The method of the invention produces α1,2-fucosyltransferasepolypeptide which are substantially pure. As used herein, the term“substantially pure” refers to a protein which is free of otherproteins, lipids, carbohydrates or other materials with which it isnormally associated. One skilled in the art can purifyα1,2-fucosyltransferase polypeptide using standard techniques forprotein purification including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.For example, the substantially pure α1,2-fucosyltransferase protein willyield a single major band of approximately 35 kD on a non-reducingpolyacrylamide gel. The purity of the α1,2-fucosyltransferasepolypeptide can also be determined by amino-terminal amino acid sequenceanalysis. α1,2-fucosyltransferase polypeptide include functionalfragments of the polypeptide, so long as biological activity remains,such as α1,2-fucosyltransferase enzymatic activity. Accordingly, theinvention includes a gene expression system and a method of producingα1,2-fucosyltransferase polypeptide which produce smaller peptidescontaining the enzymatic activity of α1,2-fucosyltransferase.

[0081] Production of α1,2-fucosyltransferase. Production ofα1,2-fucosyltransferase from the gene expression system of the inventionis achieved by culturing a gene expression system comprising a host cellrecombinantly modified with a polynucleotide encodingα1,2-fucosyltransferase polypeptide or an enzymatically active portionthereof and harvesting the α1,2-fucosyltransferase polypeptide. Themethod further comprises substantially purifying the harvestedα1,2-fucosyltransferase polypeptide using protein purification protocolswell known in the art (Current Protocols in Molecular Biology, Chapter10, eds. Ausubel, F. M. et al., 1994).

[0082] The method for producing α1,2-fucosyltransferase polypeptideinvolves culturing the gene expression system of the invention underconditions of continuous culture, such as, but not restricted to,“fed-batch cultures” or continuous perfusion cultures. Other continuousculture systems which find use in the present invention is set forth inWang, G. et al. Cytotechnology 9:41-49, 1992; Kadouri, A. et al.Advances in Animal Cell Biology and Technology for Bioprocesses, pp.327-330, Courier International, Ltd., 1989; Spier, R. E. et al.Biotechnol. Bioeng. 18:649-57, 1976.

[0083] Antibodies to α1,2-Fucosyltransferase Proteins

[0084] Antibodies that define the α1,2-fucosyltransferase gene productare within the scope of this invention, and include antibodies capableof specifically recognizing one or more α1,2-fucosyltransferase geneproduct epitopes. Such antibodies may include, but are not limited to,polyclonal antibodies, monoclonal antibodies, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection ofα1,2-fucosyltransferase gene product in a biological sample, including,but not limited to, blood, plasma, and serum. Alternatively, theantibodies may be used as a method for the inhibition of abnormalα1,2-fucosyltransferase gene product activity. Thus, such antibodies maybe utilized as part of treatment for intestinal mucosal disease, and maybe used as part of diagnostic techniques whereby patients may be testedfor abnormal levels of α1,2-fucosyltransferase gene products, or for thepresence of abnormal forms of such proteins.

[0085] For the production of antibodies against aα1,2-fucosyltransferase gene product, various host animals may beimmunized by injection with a α1,2-fucosyltransferase gene product, or aportion thereof. Such host animals may include but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsion, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG, interferon and other cytokineseffecting immunological response.

[0086] Polyclonal antibodies are a heterogenous population of antibodymolecules derived from the sera of animals immunized with an antigen,such as a α1,2-fucosyltransferase gene product, or an antigenicfunctional derivative thereof. In general, for the production ofpolyclonal antibodies, host animals such as those described above, maybe immunized by injection with α1,2-fucosyltransferase gene productsupplemented with adjuvants as also described above.

[0087] Monoclonal antibodies (mAbs), which are homogenous population ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These techniques include, but are not limited to,the hybridoma technique of Kohler and Milstein, (1975, Nature256:495-497; and U.S. Pat. No. 4,376,110), human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclinal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

[0088] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0089] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against α1,2-fucosyltransferase geneproducts. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

[0090] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclinal Fab fragments with thedesired specificity.

[0091] Methods of Detecting α1,2-Fucosyltransferase in BiologicalSamples

[0092] The antibodies described above can be used in the detection ofα1,2-fucosyltransferase polypeptides in biological samples.α1,2-fucosyltransferase polypeptide from blood or other tissue or celltype may be easily isolated using techniques which are well known tothose of skill in the art. The protein isolation methods employed hereinmay, for example, be such as those described in Harlow and Lane (Harlow,E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety.

[0093] Preferred diagnostic method for the detection of wild type ormutant α1,2-fucosyltransferase polypeptides may involve, for example,immunoassays wherein α1,2-fucosyltransferase polypeptides are detectedby their interaction with an anti-α1,2-fucosyltransferase polypeptidespecific antibody.

[0094] For example, antibodies, or fragments of antibodies, such asthose described above, useful in the present invention may be used toquantitatively or qualitatively detect the presence of wild type ormutant α1,2-fucosyltransferase polypeptides. This can be accomplished,for example, by immunofluorescence techniques employing a fluorescentlylabeled antibody coupled with light microscopic, flow cytometric, orfluorimetric detection. Such techniques are especially preferred if theα1,2-fucosyltransferase polypeptides are expressed on the cell surface.

[0095] The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof α1,2-fucosyltransferase polypeptides. In situ detection may beaccomplished by removing a histological specimen from a patient, andapplying thereto a labeled antibody of the present invention. Theantibody (or fragment) is preferably applied by overlaying the labeledantibody (or fragment) onto a biological sample. Through the use of sucha procedure, it is possible to determine not only the presence of theα1,2-fucosyltransferase polypeptide, but also its distribution in theexamined tissue. Using the present invention, those skill in the artwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

[0096] Immunoassays for wild type or mutant α1,2-fucosyltransferasepolypeptides typically comprise incubating a biological sample, such asa biological fluid, including but not limited to blood, plasma, or bloodserum, a tissue extract, freshly harvested cells, or cells which havebeen incubate in tissue culture, in the presence of a detectably labeledantibody capable of identifying α1,2-fucosyltransferase polypeptides,and detecting the bound antibody by any of a number of techniques wellknown in the art.

[0097] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling the antibodyor antibody fragments, it is possible to detect wild type or mutantα1,2-fucosyltransferase polypeptides through the use ofradioimmunoassays (RIA) (see, for example, Weintraub, Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. It is also possible to label the antibody with afluorescent compound such fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin and fluorescamine.

[0098] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu. Additionally the antibody may be detectedby coupling it to a chemiluminescent compound such as luminol,isoluminol, theramatic acreidinium ester and oxalate ester.

[0099] the following examples are intended to illustrate but not limitthe invention. While they are typical, other procedures known to thoseskilled in the art may alternatively be used to illustrate theembodiments and methods of the invention.

EXAMPLE 1 Cloning and Sequence Information of the H. pyloriFucosyltransferase (FucT) Gene

[0100] Bacterial strains and Media. H. pylori strains 26695 and UA802were used for cloning, sequencing and mutagenesis of fucT2 genes. H.pylori cells were cultured on BHI-YE agar or in BHI-YE broth undermicroaerobic conditions (Ge and Taylor, 1997, In Methods in MolecularMedicine, C. L. Clayton and H. Mobley (eds). Totowa, N.J.: Humana Press,pp. 145-152). E. coli strain DH10B was used for production ofrecombinant plasmids.

[0101] Cloning of H. pylori α(1,2)fucosyltransferase gene (Hp fucT2).Two primers, GW44 (5′-GAACACTCACACGCGTCTT-3′, position 99980-99962 inthe published H. pylori genome) and GW32 (5′-TAGAATTAGACGCTCGCTAT-3′,position 98855-98874 in the published H. pylori genome) were used to PCRamplify a 1.12 kb fragment containing Hp fucT2 from H. pylori 26695 andUA802 chromosomal DNA. In addition, by using a primer GW43(5′-CGGAGGGCTTGGGAATCAA-3′, position 99814-99796 in the published H.pylori genome ) and primer GW32, a PCR fragment of 0.96 kb carrying5′-truncated fucT2 gene was obtained from UA802 chromosomal DNA. The PCRfragments were directly cloned into pGEM-T vector (Promega) followingthe manufacturer's instructions. The orientation of the genes cloned inthe plasmids was examined by restriction enzyme analysis, and thoseclones with the fucT2 gene under the control of T7 promoter wereselected. The resultant plasmids, pGEMB3, pGEMI6, and pGEMH2, areillustrated in FIG. 3A. Subsequently, the genes cloned in the plasmidswere sequenced and were shown to be identical to the corresponding genesin the H. pylori genome.

[0102] Features of H. pylori α(1,2)fucT gene. Based on the published H.pylori genome sequence (Tomb et al., 1997, Nature 388:539-547), a pairof primers, GW44 and GW32 (FIG. 1A) were designed. These primers wereable to PCR amplify a DNA fragment (1.12 kb) from H. pylori strainUA802, which corresponds to the region containing HP0094 and HP0093 in26695. The complete nucleotide sequence of this fragment is 95%identical to that of H. pylori 26695. However, it contains a single ORFencoding a protein of 300 amino acids with a calculated molecular weightof 35,193 daltons. We designated this gene Hp fucT2 to distinguish itfrom the previously identified α(1,3)fucT which was given a name of fucT(Martin et al., 1997, J. Biol. Chem. 272:21349-21356; Ge et al., 1997,J. Biol. Chem. 272:21357-21363). Hp fucT2 gene has a unique feature inits center region. In addition to a poly C tract identified previously(Tomb et al. 1997, Nature 388:539-547, Berg et al. 1997, TrendsMicrobiol. 12:468-474), we identified a sequence of TAA repeats(imperfect, may also be GAA or AAA) immediately following the poly Csequence (FIG. 1B). The changes of the repeat number of the both tractscontribute to the variation of the fucT2 genotype (on or off status) indifferent strains (FIG. 1 A, B and Table 2).

[0103] In an attempt to find out the relationship between the fucT2 geneand the Le^(Y) phenotype, six additional H. pylori isolates wereselected for analysis (Table 2). Together with the strains 26695 andUA802, these total eight strains fall into four groups of Lewisphenotypes: Le^(X)−/Le^(Y)+, Le^(X)+/Le^(Y)+, Le^(X)+/Le^(Y)+, andLe^(X)−/Le^(Y)−. The complete nucleotide sequences of the fucT2 genesfrom these strains demonstrated an extensive variation in the poly C andTAA repeat sequence among different strains, which make the gene eitherintact (as in UA802) or franeshifted (as in 26695). Like UA802 fucT2,UA1234 fucT2 encodes an intact ORF, even though there is a deletion ofone TAA repeat. The existence of the intact fucT2 gene in UA802 andUA1234 is correlated to their Le^(Y)+ phenotype. UA1182, another examplelike 26695, contains a frameshift mutation in its fucT2 gene. Thismutation could be compensated by translation frameshifting, since adnaX-like translation frameshift cassette is present in frame.

[0104] The two strains with the Le^(X)+/Le^(Y)− phenotype, UA1174 andUA1218, displayed completely different features in their fucT2 genes. InUA1174 fucT2, there is the insertion of 2C and 2A at the hypermutableregion, resulting in a frameshift mutation. Since a dnaX-liketranslation frameshift cassette is absent (the AAAAAAG sequence is notin frame), the frameshift cannot be compensated, giving rise to aLe^(Y)− phenotype. On the other hand, UA1218 fucT2 encodes an intactORF, because the changes in the hypermutable region do not create aframeshift. However, the result from the PCR and subsequent DNAsequencing revealed a deletion of 80 bp exactly in front of the SDsequence (ribosome binding site) of UA1218 fucT2 gene. Therefore, theLe^(Y)− phenotype of UA1218 could be attributed to the absence of thepromoter for the expression of the fucT2 gene. The two strains in thelast group, UA1207 and UA1210, have an intact fucT2 gene, since thedeletion of one TAA repeat, or the change of (−C+A), respectively, doesnot create a frameshift. Therefore, the α(1,2)FucT in these two strainswould be expected to be functional. From the Le^(X)− phenotype of thesestrains we can infer that their α(1,3)FucT may be not functional, whichalso leads to the Le^(Y)− phenotype. TABLE 1 Characterization of H.pylori fucT2 mutants H. pylori ELISA reactivity (ODU) ^(b) MAbs strains^(a) anti-Le^(Y) anti-Le^(X) 26695 0.477 ± 0.047 (+) 0.414 ± 0.042 (+)26695ΔH 0.058 ± 0.014 (−) 0.437 ± 0.016 (+) 26695ΔE 0.048 ± 0.025 (−)0.829 ± 0.038 (+) UA802  0.63 ± 0.072 (+)    0 ± 0.002 (−) UA802ΔH  0.03± 0.003 (−) 0.144 ± 0.048 (+) UA802ΔE 0.069 ± 0.037 (−) 0.336 ± 0.022(+)

[0105] TABLE 2 Correlation of the fucT2 genotype with the LeY phenotypein various H. pylori strains. fucT2 genotype proposed gene status^(f)Lewis phenotype^(a) sequence translation ratio of strains LeX LeYdivergence^(b) ORF^(d) frameshift^(c) fucT fucT2 FucT/FucT2 UA802 − +reference intact − on/off on low UA1234 − + −(TAA) intact − on/off onlow 26695 + + +2C frameshifted + on off/on high UA1182 + + −Cframeshifted + on off/on high UA1174 + − +2C, +2A frameshifted − on off− UA1218 + − +C, −(AATA), intact − on off − and ΔP^(c) UA1207 − − −(TAA)intact − off on − UA1210 − − −C, +A intact − off on −

[0106] Hp fucT2 gene has a unique feature in its center region which isresponsible for the occurrence of the variant type of the gene in H.pylori 26695. It contains a poly C tract followed by imperfect TAA (orGAA, or AAA) repeats (FIG. 1B). UA802 fucT2 has a run of 12 Cs, whichallows the initiating translation frame (0 frame) be read through thisregion, giving rise to a translation product of full length. In the caseof 26695 fucT2, the existence of two more Cs (total 14 Cs) leads toearly termination of the initiating frame (HP0094) at a TGA stop codon(FIG. 1B). Downstream of HP0094, there appears to be a potential startcodon (ATG) in another frame which could be read to generate HP0093(FIG. 1A).

[0107] Since the poly C tract was identified within the Hp fucT genes(both α1,3- and α1,2 fucT), it was believed that such simpleoligonucleotide repeat regions are hypermutable and could offer anon-off mechanism for the expression of the gene (Saunders et al., 1998,Mol. Microbiol. 27:1091-1098), and may therefore be responsible for thephase variation of LPS expression. Indeed, the number of poly C repeatsin Hp fucT2 gene is variable among different strains (N=11-14, thereference UA802 fucT2 has 12 Cs, Table 2). Additionally, we observedthat the subsequent TAA repeat sequence (or called A-rich sequence) isalso a mutation hotspot. The divergence at these repetitive sequencesgave rise to the two types of the gene, encoding either a full-lengthproduct (hypothetically gene-on) or a truncated product(s)(hypothetically gene-off).

[0108] However, certain strains with a hypothetical off-status of thefucT2 gene have the Le^(Y)+ phenotype, as exemplified in 26695. Theidentification of a nucleotide sequence resembling the E. coli dnaXtranslation frameshift cassette within the 26695 fucT2 gene and theresult of in vitro expression of the gene provide a reasonable mechanismby which the full-length protein could be produced by certain off-statusfucT2 genes. Programmed translation frameshifts appear in genes from avariety of organisms and the frequency of frameshifting can be very highin some genes, approaching 100% (Farabaugh, 1996, Annu. Rev. Genet.30:507-528). The best studied −1 frameshift model is E. coli dnaX, thegene for the τ_subunit of DNA polymerase III. As a result of translationframeshifting, a truncated product (γ_subunit of DNA polymerase III) issynthesized from dnaX in a frequency of about 40%-50% (Flower andMcHenry, 1990, Proc. Natl. Acad. Sci. USA 87:3713-3717). Both τ and γsubunits are required for DNA synthesis, and are needed respectively forleading and lagging strand synthesis, due to their differentprocessivity. The main element in dnaX translation frameshift cassetteis A AAA AAG heptamer sequence in the appropriate reading frame. It hasbeen shown that the efficient frameshifting at this sequence is due tothe absence of tRNA^(Lys) with a CUU anticodon in E. coli (Tsuchihashiand Brown, 1992, Genes Dev. 6:511-519). When the tRNA^(Lys) with UUUanticodon encounters the AAG lysine codon it can easily slip to the −1frame where it interacts with the AAA lysine codon more strongly. Fromthe H. pylori whole genome sequence we know that H. pylori has only onetRNA^(Lys) with a UUU anticodon. In addition, similar to those in thednaX gene, the frameshift-stimulating elements including a putative SDsequence and a stem-loop structure were also found up- and downstream ofthe A AAA AAG sequence in 26695 fucT2 gene. Thus, it is very likely thatcertain H. pylori strains like 26695 use the same mechanism as E. colidnaX gene to generate −1 frameshift in translation of their fucT2 genes.Although we have observed the full-length and half-length protein bandsfrom the in vitro expression of 26695 fucT2 gene, the accurate frequencyof translational frameshifting in this gene, as well as in the genesfrom different stains, has not yet been determined. Also, the expressionof the gene in H. pylori cells could very well be different from thatobserved in vitro using E. coli T7 cell extract.

[0109] Unlike α(1, 3)fucosyltransferases, α(1, 2)FucTs belong to a moreheterogenous family and display very weak homology. Multiple sequencealignment for eukaryotic and prokaryotic α(1, 2)FucTs allowed us toidentify three highly conserved motifs that may have potentialstructural and/or catalytic importance. While motif I is located in theN-terminal stem region, motif II and III are located in the proposedcatalytic domain (FIG. 2). By insertion mutagenesis we showed that thedisruption of the gene at either the HindIII or EcoRI site abolishes itsfunction in the synthesis of Le^(Y), suggesting the integrity of thegene is necessary for this function.

[0110] Unexpectedly, H. pylori fucT2 mutants express more Le^(X) thanthe wild type cells. This finding suggests that Le^(X) is the substratefor Le^(Y) synthesis in H. pylori. In the commonly described mammaliansystem (Avent, 1997), Le^(Y) is produced by addition of fucose throughan α(1, 3) linkage on the H type 2 structure (FIG. 5, the pathway on theleft). However, Martin et al. (1997) found that H type 2 is not thesubstrate for Hp α(1, 3)FucT, suggesting that in the synthesis of Le^(Y)in H. pylori, α(1, 2)fucosylation may occur after α(1, 3)fucosylation(FIG. 5, the pathway on the right). Our results here are in goodagreement with this notion. Thus, disruption of α(1, 2)FucT may resultin the accumulation of its substrate, Le^(X). Furthermore, there was adifference in the Le^(X) expression between the two mutants carryingmutations at the HindIII or EcoRI site within fucT2 gene. The HindIIIsite is in the middle of fucT2 (type I) gene; downstream of it thereexists a potential small ORF corresponding to HP0093 within which theEcoRI site is located. In this ORF, which may be expressed in theHindIII mutants, there possibly remains the active site or binding site,which may compete with α(1, 3)FucT for addition of fucose to make moreLe^(X). In contrast, the binding site of α(1, 2)FucT in the EcoRImutants may be completely disrupted. Consequently, the increased Le^(X)level is more evident.

[0111] Since the synthesis of Le^(Y) requires both α(1, 3)FucT and α(1,2)FucT activities (FIG. 5), turning off either gene will give rise to aLe^(Y)− phenotype, as seen in the strains UA1174, UA1218, UA1207, andUA1210 (Table 2). If both genes are on (or partially on), the levels ofexpression of Le^(X) and Le^(Y) will depend on the ratio of theconcentrations (activities) of the two enzymes. Without knowing theactual status of α(1, 3)fucT gene (two copies), we assume that the ratioof α(1, 3)/α(1, 2)FucT in strains UA802 and UA1234 is low, so that themost (or all) of the Le^(X) synthesized by α(1, 3)FucT was converted toLe^(Y) by α(1, 2)FucT. This is supported by the observation that noLe^(X) was detected from wild type UA802, while a low level of Le^(X)was detected when the fucT2 gene was disrupted. In strains 26695 andUA1182, the fucT2 gene is in an off-on switching status due to thecompensation by translational frameshifting. As seen from the in vitroexpression of 26695 fucT2 gene and inferred from E coli dnaX gene, thefrequency of translation frameshift is presumably around 50%. Thus, theα(1, 2)FucT activity in 26695 could be lower than that in UA802. Sincethe fucT2 knock-out mutant of 26695 produced much more Le^(X) than thecorresponding UA802 mutant, we assume that the α(1, 3)FucT activity in26695 is higher than that in UA802. Taken together, we propose that theratio of α(1, 3)/α(1, 2)FucT activity in 26695 is high, which confers(Le^(X)+, Le^(Y)+) phenotype. To confirm this model, the geneticanalysis for both copies of the α(1, 3)FucT gene and comparativedetermination of the activities of both enzymes from both strains areneeded.

[0112] DNA sequencing and databases. Both strands of appropriate PCRfragments or cloned DNA fragments were sequenced using the Thermosequenase sequencing kit following the manufacturer's instructions(Amersham Life Science, Inc.). Sequence analyses were performed with theBLAST Program of the National Center of Biotechnology Information(Bethesda, Md.). The Wisconsin Package (version 9.0) of the GeneticsComputer Group (GCG) (Madison, Wis.) was used for the editing ofsequences.

[0113] Compared with the sequences in databases, Hp fucT2 has homologuesin other bacteria: a gene (wbcH) within the LPS O-antigen gene clusterof Yersinia enterocolitica serotype O:8 (Zhang et al., 1997), and a gene(epsH) within the plasmid encoded eps gene cluster essential forexopolysaccharide biosynthesis in Lactococcus lactis (van Kranenburg etal., 1997, Mol. Microbiol. 24:387-397). Although both wbcH and epsH geneproducts could act as α(1,2)FucT enzymes as predicted from sequencehomology, no experimental evidence for assignment of their function isyet available. Hp fucT2 gene product displays a low level of overallsimilarity in amino acid sequence to its eukaryotic as well asprokaryotic counterparts, with 18% identity to human Fut2 (Kelly et al.,1995, J. Biol. Chem. 270:4640-4649) and 22% identity to Y.enterocolitica WbcH. However, multiple sequence alignment revealed threeblocks of highly conserved amino acid sequences within all theα(1,2)FucTs (motifs I, II, and III in FIG. 2), which may be importantfor the enzyme function. Among them, motif II is the one proposed to bea part of the nucleotide binding domain by Breton et al. (1998,Glycobiology 8:87-94). Note that this motif is missing from the deducedamino acid sequence of 26695 fucT2 gene, because the coding region isbetween HP0094 and HP0093.

[0114] All eukaryotic α(1,2)FucTs have a typical domain structureconsisting of a short N-terminal cytoplasmic tail, a transmembranedomain, and a stem region followed by a large globular C-terminalcatalytic domain (Kleene and Berger, 1993, Biochim Biophys Acta1154:283-325). The three bacterial α(1,2)FucTs so far identified areshorter than the eukaryotic counterparts, and lack the N-terminalcytoplasmic tail and transmembrane domain (FIG. 2A). This is evidentfrom the sequence alignment and by the analysis of the secondarystructure of Hp FucT2 (hydropathy profile) which suggests it is aglobular protein without any possible transmembrane domain. TheN-terminal cytoplasmic tail and transmembrane domain of eukaryotic FucTsare proposed to have a role in Golgi localization and retention of theenzyme. The sequences of bacterial α(1,2)FucTs suggest that the enzymeis a soluble protein localized in the cytoplasm.

EXAMPLE 2 Plasmid Constructs and Expression of the H. pylori FucT Gene

[0115] Insertion mutagenesis and natural transformation. Plasmid mutantscarrying the disrupted H. pylori fucT2 gene were created by insertingthe chloramphenicol acetyltransferase (CAT) cassette (Wang and Taylor,1990, Gene 94:23-28) at HindIII or EcoRI site (FIG. 3A). Three mutantplasmids were obtained: pGHC26 (CAT cassette at HindIII site of 26695fucT2), pGEC26 (CAT cassette at EcoRI site of 26695 fucT2), and pGHC8(CAT cassette at HindIII site of UA802 fucT2). Plasmid mutants wereintroduced into the chromosome of H. pylori 26695 and UA802 by a naturaltransformation procedure. Four H. pylori fucT2 knock-out mutants wereobtained: 26695ΔH, 26695ΔE, 802ΔH, and 802ΔE (Note: There is no EcoRIsite in UA802 fucT2 gene, and 802ΔE was obtained by transforming pGEC26into UA802.)

[0116] In vitro expression of Hp fucT2 gene. The plasmids containingHpfucT2 genes under the control of the T7 promoter, as well as thoseplasmids with CAT cassette insertion within the fucT2 genes, werepurified by CsCl gradient ultracentrifugation. The purified supercoiledcircular DNA were used as template for in vitro expression of the clonedgenes using E. coli T7 S30 Extract System (Promega) following themanufacturer's instruction. The expressed gene products were labeledwith [³⁵S] methionine and analyzed on 0.1% SDS-12% polyacrylamide gelfollowed by autoradiography.

[0117] Immunoelectron microscopy. H. pylori broth cultures were absorbedonto Formvar-coated electron microscope grids and washed in phosphatebuffer. The samples were incubated with primary anti-Le^(Y) MAb isotypeIgM (Signet Laboratories, Inc.) and further incubated with goatanti-mouse IgM-10 nm colloidal gold conjugate (EY Laboratories, Inc.,San Mateo, Calif.). Positive labeling was determined by the presence ofgold particles on unfixed and unstained H. pylori cells.

[0118] ELISA with H. pylori whole cell suspensions. The primaryantibodies used were anti-Le^(X)(mAB BG-7) and anti-Le^(Y)(mAB BG-8)(Signet Laboratories Inc. Dedham, Mass.). The secondary antibody wasanti-mouse IgG+IgM conjugated to horse-radish peroxidase (HRP) (Biocan#115 035 068, Mississauga, Ontario) diluted 1:2000. The reaction wasstopped with 4 mM sodium azide and the absorbance was recorded at 405 nmusing a Titretek Multiscan MC (Helsinki, Finland) microtitre platereader.

[0119] SDS-PAGE and immunoblot analysis of H. pylori LPS. Proteinase Ktreated whole cells extracts of H. pylori strains were prepared andsubjected to electrophoresis on a stacking gel of 5% acrylamide and aseparating gel of 15% acrylamide. LPS on the gel was detected either bysilver staining or by immunoblotting. The LPS transferred tonitrocellulose membrane (pore size 0.22 μm, Micron Separations Inc.Westboro Mass.) were probed with anti-Lewis structure antibodies (1:100dilution), and subsequently with goat anti-mouse antibody conjugated tohorse radish peroxidase (1:2000 dilution). Blots were developed using anenhanced chemiluminescence kit (Amersham) according to themanufacturer's specifications.

[0120] Both types of Hp fucT2 gene produce a full-length protein invitro.

[0121] As illustrated in FIG. 3A and described in ExperimentalProcedures, Hp fucT2 genes were amplified by PCR and cloned into thepGEM-T vector under the control of a T7 promoter. The genes cloned intothe plasmids are identical to those original genes in the H pylorigenome as verified by DNA sequencing. The plasmid pGEMB3 contains 26695fucT2 gene; pGEMH2 contains a 5′-truncated 802 fucT2 gene; and pGEMI6contains complete 802 fucT2 gene. Subsequently, the CAT cassette wasinserted at the HindIII or EcoRI site within the cloned Hp fucT2 genesto obtain plasmid mutants pGHC26 (CAT cassette at HindIII site of 26695fucT2), pGEC26 (CAT cassette at EcoRI site of 26695 fucT2), and pGHC8(CAT cassette at HindIII site of UA802 fucT2). The six plasmid DNAs wereused as templates for in vitro transcription-translation assays toexamine the protein products encoded by the corresponding genes.

[0122] The expressed protein products analyzed on SDS-PAGE are shown inFIG. 3B. The expression of 802 fucT2 gene (pGEMI6, lane 5) gave rise toa major protein of 33 KD, which is very close to that expected from thededuced aa sequence (35 KD). Three weak bands with smaller molecularweights may result from translations starting at internal ATG codons orfrom degradation of the full length protein. As expected, 5′-truncated802 fucT2 did not produce the full-length protein (pGEMH2, lane 4). Forthe expression of 26695 fucT2 (pGEMB3, lane 3), two small proteins of 18and 13 KD were expected based on the DNA sequence of the gene. However,in addition to a 17 KD protein band which may represent the half-lengthgene product (HP0094), we observed a full-length (33 KD) protein band.To confirm that this result was not due to a mutation in the clonedgene, the sequence of the actual plasmid DNA (pGEMB3) used for the invitro transcription-translation assay was re-examined, and no change wasfound compared to the original 26695 fucT2 gene.

[0123] The observation that 26695 fucT2 gene produces the full-lengthprotein prompted us to consider other possibilities which could accountfor this result: RNA polymerase slippage in transcription or ribosomeslippage in translation. By re-examining the DNA sequences of the simplerepeat region within Hp fucT2 gene (FIG. 1B), we found three motifs (XXXY YYZ) typical of programmed translation frameshift (Farabaugh, 1996)occurred in the appropriate reading frame. The first one (C CCT TTA),located upstream of the poly C tract, exists in 26695 fucT2, but not in802 fucT2. The second one (A AAA AAG), located downstream of the poly Ctract, is present in the reading frame of 26695 fucT2, but not in thereading frame of 802 fucT2. This motif is identical to the extremelyslippery heptanucleotide found in the mRNA of E. coli dnaX(Flower andMcHenry, 1990). Other elements of dnaX frameshifting signal (Larsen etal., 1994, J. Bacteriol. 176:6842-6851) including an upstream SDsequence and a downstream stem-loop structure which serve as stimulatorsof the frameshifting are also present in the deduced 26695 fucT2 mRNAsequence (FIG. 1C). Therefore, A similar mechanism for −1 frameshift asin dnaX is very likely at work in 26695 fucT2: exactly at the sitebehind the poly-C region where a frameshift has occurred (relative tothe prototype 802 fucT2) and before-encountering the stop codon, thereading frame could be shifted back (at a certain frequency) to thereading frame of the prototype gene, so that a full-length protein couldbe produced. Interestingly, just four codons before the HP0093 startcodon, there exists another A AAA AAG sequence, both in 26695 and 802fucT2 genes (FIG. 1B, line 4). However, no upstream SD sequence anddownstream stem-loop structure were found around this slippery sequence.

[0124] Analysis of the fucT2 genes from several different strains (Table2) demonstrated the various factors affecting expression of this geneand the ultimate Lewis phenotype. First, some divergence in the promoterregion was observed among different strains, which could contribute tothe differential expression of the gene through regulatingtranscription. Although the function of the promoter of Hp fucT2 genewas not performed in detail, apparently in strain UA1218 the promoterwas completely missing resulting in the off-status of the gene. Second,two elements within the coding region of the gene were identified thatcould affect the coding ability of the gene. The first element, thesimple sequence repeat region, is a mutation hotspot. As suggestedpreviously (Tomb et al. 1997, Berg et al. 1997, Saunders et al. 1998),the frameshift mutation produced by DNA polymerase slippage during thereplication of the gene may provide a mechanism for the switchingbetween on and off status of the gene (at a frequency of <1%), whichcould account for the phase variation of Le^(Y) expression reported byAppelmelk et al. (1998, Infect. Immun. 66:70-76). The extensive sequencedivergence at this hypermutable region among various strains and theresulting two types (intact or frameshifted) of the gene support thenotion that this strand-slippage mechanism occurs in H. pylori.

[0125] The second element within the gene is the slippery sequence forribosome translation which is located immediately behind thehypermutable region. In certain strains that have a −1 frameshiftmutation (relative to the prototype), such as 26695 and UA1182, thetranslation could be shifted back to the prototype reading frame at ahigh frequency, producing functional proteins. In other strains thathave a prototype reading frame (e.g. UA802) or +1 frameshift (e.g.UA1174), this slippery sequence is not in frame, thus is not functional.Therefore, the frameshift mutation in UA1174 fucT2 cannot be compensatedat the translation stage, resulting in the off-status of the gene.Interestingly, in the reading frame of the prototype fucT2 genes such asthat of UA802 there exists another A AAA AAG slippery sequence in framebut without enhancing elements (FIG. 1B, line 4). It is not knownwhether translational frameshifting occur here at very low frequency toproduce a minor fraction of truncated protein. If so, it could affectthe level of the Le^(Y) production, although insignificantly. Insummary, it is propose that translational frameshifting may offer H.pylori an mechanism by which the full-length (active) and truncated(inactive or less efficient) enzymes can be produced in various ratioswhich account for the different levels of Le^(Y) production amongvarious strains. This ratio could also be influenced by certainenvironmental factors in the course of H. pylori-host interaction,leading to the varied level of Le^(Y) expression in an individualstrain.

[0126] Effect of fucT2 Knock-Out Mutations on the Expression of Le^(Y)and Le^(X) in H. pylori.

[0127] To demonstrate the requirement of Hp fucT2 in the biosynthesis ofLe^(Y), we performed insertion mutagenesis of fucT2. As described inExperimental Procedures, we constructed four H. pylori fucT2 knock-outmutants: 26695ΔH, 26695ΔE, 802ΔH, and 802ΔE, in which the fucT2 gene ofH. pylori 26695 or UA802 was disrupted by insertion of a CAT cassette atHindIII or EcoRI site, respectively. The insertion of the CAT cassetteat the specific location of the fucT2 gene in the H. pylori genome wasverified by PCR amplification of an expected fragment and by DNAsequencing of the region surrounding the insertion site. The phenotypesof these H. pylori mutants for Le^(Y) expression were examined byelectron microscopy and by ELISA.

[0128]FIG. 4 shows an example of the transmission electron micrographsof UA802 wild type and mutant cells immunostained with anti-Le^(Y)MAb.Wild type cells strongly express Le^(Y), as evidenced by the presence ofmany gold particles. In contrast, the mutant cells, 802ΔH and 802ΔE(shown here is only 802_ΔH), were negative for immunogold labeling usinganti-Le^(Y) antibody. A similar pattern of electron micrographs forstrain 26695 (Le^(Y) positive) and its mutants (Le^(Y) negative) wasobserved.

[0129] A quantitative examination for the expression of Le^(Y) as wellas Le^(X) by these strains detected by ELISA is given in Table 1. Wildtype strain 26695 expresses both Le^(Y) and Le^(X) (ODU=0.48 and 0.41,respectively), while wild type UA802 strongly expresses Le^(Y)(ODU=0.63) but no Le^(X). All of their isogenic mutants were negativefor Le^(Y) (ODU<0.1), suggesting that disruption of the fucT2 gene atboth HindIII and EcoRI site abolish Le^(Y) expression. Interestingly,there is an increase in the expression of Le^(X) for the fucT2 mutants,especially when the mutation is at the EcoRI site.

[0130] Further characterization of these mutants was carried out bySDS-PAGE and immunoblots of the LPS for detection of Le^(Y) and Le^(X)(FIG. 5). Silver stained gels revealed no change in the LPS side chainlength for all the mutants compared with the wild type cells. Theimmunoblots confirmed that Le^(Y) is expressed by the wild type strains26695 and UA802, and is no longer expressed in all the fucT2 mutantstrains (FIG. 5A). Wild type UA802 does not express any Le^(X) on itssurface, but its isogenic fucT2 mutants do express Le^(X) (FIG. 5B).There was no significant difference on the Le^(X) expression levelsbetween the two mutants (802ΔH and 802ΔE), which is different from theELISA results. Since there is Le^(X) expression in the wild type strain26695, the increase of Le^(X) expression in its mutant strains is not soevident. Similar to the ELISA results, however, a significant increasein Le^(X) expression was observed in 26695ΔE, but not in 26695ΔH.

EXAMPLE 3 Enzymatic Activities of H. pylori α1,2 Fucosyltransferase

[0131] Overexpression of the H. pylori fucosyltransferase in E. coli. Ina typical experiment, E. coli CLM4 (pGP1-2) cells haboring a plasmidcarrying an H. pylori fucT gene (pBKHp763fucT39, pGEMH2, pGEMI6 orpGEMB3) were grown in 25 ml liquid LB medium with appropriateantibiotics (kanamycin and ampicillin) at 30° C. to an optical densityof 0.5-0.7 at 600 nm. After being collected, the cells were washed oncewith M9 medium, resuspended in 5 ml of supplemented M9 medium, andfurther incubated at 30° C. for 1 h. To induce the expression of thefucT gene, the cell culture was shifted to 42° C. by adding 5 mlprewarmed (55° C.) supplemented M9 medium. After incubation at 42° C.for 15 min, rifampicin was added to a final concentration of 200 μg/ml,and cell growth was continued at 42° C. for 20 min.

[0132] For analysis of the protein on SDS-PAGE, a small aliquot (0.5 ml)of the cell culture was taken, and 2.5 μl of [³⁵S]-methionine (4.35×10¹³Bq/mmol, 3.7×10⁸ Bq/ml, NEN™, Boston, Mass.) was added. After furthergrowth at 30° C. for 30 min, the cells were harvested, resuspended in100 μl sample buffer (50 mM Tris-HCl, pH6.8, 1% (w/v) SDS, 20 mM EDTA,1% (v/v) mercaptoethanol, 10% (v/v) glycerol), and boiled for 3 minbefore loading on to the gel. For the preparation of the sample for theenzyme assay, the remaining part (major aliquot, 9.5 ml) of the cellculture after induction was further incubated at 30° C. for 30 min, thenharvested. The cells were washed with 1.5 ml of 20 mM HEPES (pH 7.0),and resuspended in 1.5 ml of this buffer supplemented with 0.5 mM PMSF.

[0133] Preparation of cell lysates or cell extracts for thefucosyltransferase assay. The E. coli cells containing overproduced HpFucT proteins, which were in HEPES buffer with PMSF as described above,were disrupted with a French press at 7000 lb/in² at 4° C. The celllysates were used directly for enzyme assays. For determining thelocation of the enzyme activities, the cytoplasmic and membranefractions were separated as follows. The cell lysates were centrifugedat 13,000 xg at 4° C. for 10 min. The cell debris were discarded and thesupernatant was subjected to ultracentrifugation at 128,000 xg (BeckmanTL100/rotor 100.2) at 4° C. for 1 h. The supernatant was collected asthe cytoplasmic fraction. The membrane pellets were resuspended in asmall volume of the same buffer and treated with 1 M NaCl.

[0134] Fucosyltransferase assay. Assays of Hp α1,2 and α1,3 FucTactivities were carried out according to the method described by Chan etal. (1995, Glycobiology 5:683-688) with some modifications. Reactionswere conducted at 37° C. for 20 min in a volume of 20 μl containing 1.8mM acceptor, 50 μM GDP-fucose, 60000 dpm GDP-[³H]fucose, 20 mM HEPESbuffer (pH7.0), 20 mM MnCl₂, 0.1 M NaCl, 35 mM MgCl₂, 1 mM ATP, 5 mg/mlBSA, and 6.2 μl of the enzyme preparation. The acceptors used in thisstudy were: LacNAc [βGal 1-4 βGlcNAc], Le^(X) [βGal 1-4 (α Fuc1-3)βGlcNAc], Type 1 [βGal 1-3 βGlcNAc], and Le^(a) [βGal 1-3 (α Fuc1-4)βGlcNAc]. GDP-[³H]fucose (1.9×10¹¹ Bq/ml/mmol) was obtained fromAmerican Radiolabeled Chemicals Inc. (St. Louis, Mo.). Sep-Pak Plus C-18reverse-phase cartridges were purchased from Waters (Mississauga, ON).For calculation of the specific activity of the enzyme (micro-units permilligram protein), protein concentrations of the cell extracts weredetermined with a BCA protein assay kit (Pierce, Rockford, Ill.) usingBSA as a standard according to the supplier's instructions.

[0135] Acceptor specificity of Hp α1,2 FucT. Plasmid pGEMI6 carries theprototype fucT2 gene from H. pylori UA802 which produces a high level ofLe^(Y). Initially, we quantitated the α1,2 FucT activity by using LacNAcand Le^(X) as acceptors, the two potential substrates of α1,2 FucT forthe synthesis of Le^(Y) (FIG. 5). Surprisingly, almost no activity wasdetected using LacNAc as an acceptor, whereas considerable activity wasobserved for the monofucosylated Le^(X) acceptor (Table 3B). Thespecific activity of α1,2 FucT is much lower compared to that of α1,3FucT (Table 3A).

[0136] In mammalian cells, the same α1,2 FucT enzyme (H or Se,tissue-specific) is normally responsible for the synthesis of both Htype 1 and H type 2 structures (Sarnesto et al., 1990, J. Biol. Chem.265:15067-15075; Sarnesto et al., 1992, J. Biol. Chem. 267:2732-2744).To determine whether the Hpα1,2 FucT is also involved in the synthesisof Le^(b), we measured its activity with type 1 oligosaccharideacceptors (Table 3B). Even though UA802 does not express type 1 Lewisantigen, its α(1,2) FucT enzyme can transfer fucose to Type 1 and Le^(a)acceptors. Compared to Le^(X), type 1 and Le^(a) are even more efficientsubstrates for Hp α1,2 FucT (2-fold more active). Thus, Hp α1,2 FucT canalso synthesize H type 1 and Le^(b). TABLE 3 Activity and acceptorspecificity of H. pylori fucosyltransferases Overexpressed specificprotein^(a) proposed activity relative (plasmid construct) acceptorproduct (μU/mg)^(b) activity (%)^(c) A α1,3 FucT LacNAc Le^(X) 1480(pBKHp763fucT39) B α1,2 FucT (UA802) LacNAc H type 2 14 ± 8 4.5 (pGEMI6)Le^(X) Le^(Y) 150 ± 33 49 Type 1 H type 1 309 ± 28 100 Le^(a) Le^(b) 301± 13 97

[0137] Analysis of the Reaction Products of Hp α1,2 FucT by CapillaryElectrophoresis.

[0138] The reaction products synthesized from different acceptors by theHp α1,2 FucT were further characterized by capillary electrophoresiswith laser-induced fluorescence detection. The reaction mixturecontained the overproduced UA802 α1,2 FucT protein (from pGEMI6 clone),GDP-fucose, and different acceptors labeled with tetramethylrhodamine(TMR). The results (FIG. 7) confirmed the data from the enzyme assayusing radioactive labeled GDP-fucose (Table 3B) by identifying theproducts of the reactions.

[0139] When using LacNAc as an acceptor (FIG. 7A, line a), no reactionproduct representing H type 2 was observed, suggesting that LacNAc isnot a substrate for Hp α1,2 FucT. In the reaction using Le^(X) as anacceptor (FIG. 7A, line b), a small new peak was produced, whichco-migrated with a synthetic Le^(Y)-TMR (standard Le^(Y)) in theelectropherogram, indicating that this new peak represents the Le^(Y)product synthesized from Le^(X) by Hp α1,2 FucT. Similarly, by usingType 1 or Le^(a) as acceptors (FIG. 7B), new peaks co-migrating withauthentic products, H type 1 or Le^(b) respectively, were observed. Asnegative controls, the protein extract from the E. coli CLM4 (pGP1-2)clone containing the pGEM vector without Hp fucT2 gene was used in thereactions for each acceptor tested above, and no peaks for the productsof α1,2 FucT were observed.

[0140] Novel α1,2 fucosyltransferase. Determination of activities of theresponsible fucosyltransferases is direct proof to distinguishingbetween the two possible pathways (FIG. 5). The observation in thisstudy that Le^(X) but not LacNAc is the substrate for the Hp α1,2 FucTclearly indicated that H. pylori prefers to use the Le^(X) pathway tosynthesize Le^(Y) (FIG. 8A). Other supporting evidence came from theenzyme assay for Hp α1,3 FucT: (I) LacNAc is an excellent substrate forHp α1,3 FucT (Ge et al., 1997; Martin et al., 1997; and Table 3A); and(ii) Martin et al. (1997) found that H type 2 was not the substrate ofan Hp α1,3 FucT. It should be noted, however, that thefucosyltransferases from different H. pylori strains may have differentacceptor specificity. Further studies on combined analysis of the α1,3and α1,2 FucTs from various H. pylori strains are needed to elucidatewhether this novel pathway for the synthesis of Le^(Y) is general in H.pylori or is strain-specific.

[0141] In addition to its function in Le^(Y) synthesis, Hp α1,2 FucT isalso active on type 1 Lewis structures (summarized in FIG. 8B). Thisprovides a basis for the recent finding that Type 1 (Le^(c)), H type 1,and Le^(a) are expressed in certain H. pylori strains (Le^(b) was alsodetected in some strains by serological methods but has not yet beenconfirmed by structural analysis) (Monteiro et al., 1998, J. Biol. Chem.273:11533-11543). Here again, the activity of the Hp α1,2 FucT tosynthesize Le^(b) from Le^(a) indicated that this bacterial enzyme isdifferent from the normal mammalian counterparts which cannot use Le^(a)as substrate. To know if Le^(b) can be synthesized from H type 1 in H.pylori awaits the detection of an α1,4 FucT. The α1,2 FucT characterizedin this study is from H. pylori strain UA802 which does not produce anytype 1 Lewis antigen. This suggests that the same α1,2 FucT enzyme couldbe used in the strains that produce type 1 epitopes. The failure toproduce type 1 Lewis antigens in many H. pylori strains could be due tothe inavailability of one of the other enzymes involved in the synthesisof Lewis antigens such as galactosyltransferase that adds βGal to GlcNAcor α1,3/4 FucT that places the αFuc unit at βGlcNAc.

[0142] In summary, in contrast to the normal mammalian α1,2 FucT (H orSe enzyme), Hp α1,2 FucT prefers to use Lewis X [βGal 1-4 (α Fuc1-3)βGlcNAc] rather than LacNAc [βGal 1-4 βGlcNAc] as a substrate,suggesting that H. pylori uses a novel pathway (via Lewis X) tosynthesize Lewis Y. Hp α1,2 FucT also acts on type 1 acceptor [βGal 1-3βGlcNAc] and Lewis a [βGal 1-3 (α Fuc1-4) βGlcNAc], which provides H.pylori with the potential to synthesize H type 1 and Lewis b epitopes.The ability to transfer fucose to a monofucosylated substrate (Lewis Xor Lewis a) makes Hp α1,2 FucT distinct from normal mammalian α1,2 FucT.

[0143] Hp α1,2 FucT is a soluble protein. DNA sequence analysispredicted the Hp α1,2 FucT to be a hydrophilic protein, and the same istrue for Hp α1,3 FucT (Ge et al., 1997). However, the determination ofHp α1,3 FucT activity from the overexpressed proteins demonstrated thatthe majority of the activity were present in the membrane fraction (Geet al., 1997). To delineate the cellular location of the Hp α1,2 FucTactivity, cytoplasmic and membrane fractions of E. coli cellsoverproducing Hp α1,2 FucT proteins were prepared as described inMaterials and Methods. The activity in both fractions was determinedusing Le^(X) or Type 1 as acceptors (Table 4). There was no detectableactivity in the membrane fraction when using Le^(X) as an acceptor. Byusing Type 1 as an acceptor, a very low amount of activity (negligible)was detected in the membrane fraction, which accounts for less than 3%of the total activity. These results indicated that Hp α1,2 FucT is asoluble cytoplasmic protein. TABLE 4 Enzyme activities of H. pylori α1,2FucT in cytoplasmic and membrane fractions. Acceptor specific activityExp. No. used protein fraction^(a) (μU/mg protein) total activity(μU)^(b) relative activity (%)^(c) 1 Le^(x) cytoplasm 38 49  100membrane  0 0  0 2 Le^(x) cytoplasm 41 54  100 membrane  0 0  0 Type 1cytoplasm 78 108  100 membrane  8 3  3

[0144]

1 23 1 1119 DNA Helicobacter pylori CDS (137)...(1036) 1 gaacactcacacgcgtcttt ttcaaataaa aaattcaaat gatttgaaag cgttacccca 60 ctttttaggcttttattgaa aaagggcttt aaagttggct aaaataggcg ttttatttga 120 aaaacaaaggggttga atg gct ttt aaa gtg gtg caa att tgt ggg ggg ctt 172 Met Ala PheLys Val Val Gln Ile Cys Gly Gly Leu 1 5 10 ggg aat caa atg ttt caa tacgct ttc gct aaa agt ttg caa aaa cac 220 Gly Asn Gln Met Phe Gln Tyr AlaPhe Ala Lys Ser Leu Gln Lys His 15 20 25 ctt aat acg ccc gtg cta tta gacact act tct ttt gat tgg agc aat 268 Leu Asn Thr Pro Val Leu Leu Asp ThrThr Ser Phe Asp Trp Ser Asn 30 35 40 agg aaa atg caa tta gag ctt ttc cctatt gat ttg ccc tat gcg aat 316 Arg Lys Met Gln Leu Glu Leu Phe Pro IleAsp Leu Pro Tyr Ala Asn 45 50 55 60 gca aaa gaa atc gct ata gct aaa atgcaa cat ctc ccc aag tta gta 364 Ala Lys Glu Ile Ala Ile Ala Lys Met GlnHis Leu Pro Lys Leu Val 65 70 75 aga gat gca ctc aaa tac ata gga ttt gatagg gtg agt caa gaa atc 412 Arg Asp Ala Leu Lys Tyr Ile Gly Phe Asp ArgVal Ser Gln Glu Ile 80 85 90 gtt ttt gaa tac gag cct aaa ttg tta aag ccaagc cgt ttg act tat 460 Val Phe Glu Tyr Glu Pro Lys Leu Leu Lys Pro SerArg Leu Thr Tyr 95 100 105 ttt ttt ggc tat ttc caa gat cca cga tat tttgat gct ata tcc tct 508 Phe Phe Gly Tyr Phe Gln Asp Pro Arg Tyr Phe AspAla Ile Ser Ser 110 115 120 tta atc aag caa acc ttc act cta ccc ccc cccccc gaa aat aat aaa 556 Leu Ile Lys Gln Thr Phe Thr Leu Pro Pro Pro ProGlu Asn Asn Lys 125 130 135 140 aat aat aat aaa aaa gag gaa gaa tac cagcgc aag ctt tct ttg att 604 Asn Asn Asn Lys Lys Glu Glu Glu Tyr Gln ArgLys Leu Ser Leu Ile 145 150 155 tta gcc gct aaa aac agc gta ttt gtg catata aga aga ggg gat tat 652 Leu Ala Ala Lys Asn Ser Val Phe Val His IleArg Arg Gly Asp Tyr 160 165 170 gtg ggg att ggc tgt cag ctt ggt att gattat caa aaa aag gcg ctt 700 Val Gly Ile Gly Cys Gln Leu Gly Ile Asp TyrGln Lys Lys Ala Leu 175 180 185 gag tat atg gca aag cgc gtg cca aac atggag ctt ttt gtg ttt tgc 748 Glu Tyr Met Ala Lys Arg Val Pro Asn Met GluLeu Phe Val Phe Cys 190 195 200 gaa gac tta aaa ttc acg caa aat ctt gatctt ggc tac cct ttc acg 796 Glu Asp Leu Lys Phe Thr Gln Asn Leu Asp LeuGly Tyr Pro Phe Thr 205 210 215 220 gac atg acc act agg gat aaa gaa gaagag gcg tat tgg gat atg ctg 844 Asp Met Thr Thr Arg Asp Lys Glu Glu GluAla Tyr Trp Asp Met Leu 225 230 235 ctc atg caa tct tgc aag cat ggc attatc gct aat agc act tat agc 892 Leu Met Gln Ser Cys Lys His Gly Ile IleAla Asn Ser Thr Tyr Ser 240 245 250 tgg tgg gcg gct tat ttg atg gaa aatcca gaa aaa atc att att ggc 940 Trp Trp Ala Ala Tyr Leu Met Glu Asn ProGlu Lys Ile Ile Ile Gly 255 260 265 ccc aaa cac tgg ctt ttt ggg cat gaaaat att ctt tgt aag gaa tgg 988 Pro Lys His Trp Leu Phe Gly His Glu AsnIle Leu Cys Lys Glu Trp 270 275 280 gtg aaa ata gaa tcc cat ttt gag gtaaaa tcc caa aaa tat aac gct 1036 Val Lys Ile Glu Ser His Phe Glu Val LysSer Gln Lys Tyr Asn Ala 285 290 295 300 taaagcggct taaaaaaagg gcttactagaggtttaatct ttgattttag atcggatttc 1096 tttatagcga gcgtctaatt cta 1119 2300 PRT Helicobacter pylori 2 Met Ala Phe Lys Val Val Gln Ile Cys GlyGly Leu Gly Asn Gln Met 1 5 10 15 Phe Gln Tyr Ala Phe Ala Lys Ser LeuGln Lys His Leu Asn Thr Pro 20 25 30 Val Leu Leu Asp Thr Thr Ser Phe AspTrp Ser Asn Arg Lys Met Gln 35 40 45 Leu Glu Leu Phe Pro Ile Asp Leu ProTyr Ala Asn Ala Lys Glu Ile 50 55 60 Ala Ile Ala Lys Met Gln His Leu ProLys Leu Val Arg Asp Ala Leu 65 70 75 80 Lys Tyr Ile Gly Phe Asp Arg ValSer Gln Glu Ile Val Phe Glu Tyr 85 90 95 Glu Pro Lys Leu Leu Lys Pro SerArg Leu Thr Tyr Phe Phe Gly Tyr 100 105 110 Phe Gln Asp Pro Arg Tyr PheAsp Ala Ile Ser Ser Leu Ile Lys Gln 115 120 125 Thr Phe Thr Leu Pro ProPro Pro Glu Asn Asn Lys Asn Asn Asn Lys 130 135 140 Lys Glu Glu Glu TyrGln Arg Lys Leu Ser Leu Ile Leu Ala Ala Lys 145 150 155 160 Asn Ser ValPhe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly 165 170 175 Cys GlnLeu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala 180 185 190 LysArg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Lys 195 200 205Phe Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Thr Asp Met Thr Thr 210 215220 Arg Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser 225230 235 240 Cys Lys His Gly Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp AlaAla 245 250 255 Tyr Leu Met Glu Asn Pro Glu Lys Ile Ile Ile Gly Pro LysHis Trp 260 265 270 Leu Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp ValLys Ile Glu 275 280 285 Ser His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala290 295 300 3 19 DNA Artificial Sequence synthetically generatedoligonucleotide 3 gaacactcac acgcgtctt 19 4 20 DNA Artificial Sequencesynthetically generated oligonucleotide 4 tagaattaga cgctcgctat 20 5 19DNA Artificial Sequence synthetically generated oligonucleotide 5cggagggctt gggaatcaa 19 6 231 DNA Helicobacter pylori 6 tatatcccctttaatcaagc aaaccttcac tctacccccc ccccccccga aaataataag 60 aataataataaaaaagagga agaatatcag tgcaagcttt ctttgatttt agccgctaaa 120 aacagcgtgtttgtgcatat aagaagaggg gattatgtgg ggattggctg tcagcttggt 180 attgactatcaaaaaaaggc gcttgagtat atggcaaagc gtgccaaaca t 231 7 230 DNA Helicobacterpylori 7 tatatcctct ttaatcaagc aaaccttcac tctacccccc ccccccgaaaataataaaaa 60 taataataaa aaagaggaag aataccagcg caagctttct ttgattttagccgctaaaaa 120 cagcgtattt gtgcatataa gaagagggga ttatgtggga ttggctgtcagcttggtatt 180 gattatcaaa aaaaggcgct tgagtatatg gcaaagcgcg tgccaaacat230 8 60 RNA Helicobacter pylori 8 uaauaagaau aauaauaaaa aagaggaagaauaucagugc aagcuuucuu ugauuuuagc 60 9 11 PRT Homo sapiens 9 Gly Arg PheGly Asn Gln Met Gly Gln Tyr Ala 1 5 10 10 11 PRT Homo sapiens 10 Gly ArgLeu Gly Asn Gln Met Gly Glu Tyr Ala 1 5 10 11 11 PRT Helicobacter pylori11 Gly Gly Leu Gly Asn Gln Met Phe Gln Tyr Ala 1 5 10 12 11 PRT Yersiniaenterocolitica 12 Gly Gly Leu Gly Asn Gln Leu Phe Gln Val Ala 1 5 10 1311 PRT Lactococcus lactis 13 Gly Asn Leu Gly Asn Gln Leu Phe Ile Tyr Ala1 5 10 14 11 PRT Homo sapiens 14 Val Gly Val His Val Arg Arg Gly Asp TyrLeu 1 5 10 15 11 PRT Homo sapiens 15 Val Gly Val His Val Arg Arg Gly AspTyr Val 1 5 10 16 11 PRT Helicobacter pylori 16 Val Phe Val His Ile ArgArg Gly Asp Tyr Val 1 5 10 17 11 PRT Yersinia enterocolitica 17 Val GlyIle His Ile Arg Arg Gly Asp Phe Val 1 5 10 18 11 PRT Lactococcus lactis18 Ile Cys Val Ser Ile Arg Arg Gly Asp Tyr Val 1 5 10 19 10 PRT Homosapiens 19 Gly Thr Phe Gly Phe Trp Ala Ala Tyr Leu 1 5 10 20 10 PRT Homosapiens 20 Gly Thr Phe Gly Ile Trp Ala Ala Tyr Leu 1 5 10 21 10 PRTHelicobacter pylori 21 Ser Thr Tyr Ser Trp Trp Ala Ala Tyr Leu 1 5 10 2210 PRT Yersinia enterocolitica 22 Ser Thr Phe Ser Trp Trp Ala Ala IleLeu 1 5 10 23 10 PRT Lactococcus lactis 23 Ser Ser Phe Ser Trp Trp ThrGlu Phe Leu 1 5 10

What is claimed is:
 1. A substantially purified α1,2-fucosyltransferase.2. The substantially purified α1,2-fucosyltransferase of claim 1,wherein the polypeptide catalyzes the synthesis of Lewis Y.
 3. Thepolypeptide of claim 1, wherein the polypeptide lacksα1,4-fucosyltransferase activity.
 4. The polypeptide of claim 1, whereinthe polypeptide lacks α1,3-fucosyltransferase activity.
 5. Thepolypeptide of claim 1, wherein the polypeptide lacksα1,4-fucosyltransferase and α1,3-fucosyltransferase activity.
 6. Thepolypeptide of claim 1, wherein the polypeptide has an amino acidsequence comprising SEQ ID NO:
 2. 7. An isolated polynucleotide encodingthe polypeptide of claim
 1. 8. The polynucleotide of claim 7, whereinthe sequence encodes the amino acid sequence having SEQ ID NO:
 2. 9. Thepolynucleotide of claim 8, comprising a sequence having at least onerepeat of the sequence X XXY YYZ, wherein X=A or C, Y=A or T and Z=A orG.
 10. A polynucleotide selected from the group consisting of: a) SEQ IDNO:1; b) SEQ ID NO: 1, wherein T is U; c) nucleic acid sequencescomplementary to a) or b); and d) fragments of a), b), or c) that are atleast 15 nucleotide bases in length and that hybridize to DNA whichencodes the polypeptide set forth in SEQ ID NO:2.
 11. A vectorcontaining the polynucleotide of claim
 7. 12. A host cell containing thevector of claim
 11. 13. An antibody which selectively binds to thepolypeptide of claim
 1. 14. The antibody of claim 13, wherein theantibody is monoclonal.
 15. The antibody of claim 13, wherein theantibody is polyclonal.
 16. A method for detectingα1,2-fucosyltransferase polypeptide in a sample, comprising: a)contacting the sample with the antibody of claim 13; and b) detectingbinding of the antibody to α1,2-fucosyltransferase polypeptide, whereinbinding is indicative of the presence of α1,2-fucosyltransferasepolypeptide in the sample.
 17. The method of claim 16, wherein thesample is tissue.
 18. The method of claim 16, wherein the sample is abiological fluid.
 19. The method of claim 16, wherein the presence ofα1,2-fucosyltransferase polypeptide in the sample is indicative ofinfection by Helicobacter pylori.
 20. The method of claim 16, whereinthe presence of α1,2-fucosyltransferase polypeptide in the sample isindicative of the presence of malignant cells.
 21. A method fordetecting α1,2-fucosyltransferase polynucleotide in a sample,comprising: a) contacting a sample suspected of containingα1,2-fucosyltransferase polynucleotide with a nucleic acid probe thathybridizes to α1,2-fucosyltransferase polynucleotide; and b) detectinghybridization of the probe with α1,2-fucosyltransferase polynucleotide,wherein-the detection of hybridization is indicative ofα1,2-fucosyltransferase polynucleotide in the sample.
 22. The method ofclaim 21, wherein the nucleic acid probe is selected from the groupconsisting of: a) a nucleic acid sequence set forth in SEQ ID NO: 1; b)a nucleic acid sequence set forth in SEQ ID NO; 1, wherein T is U; c) anucleic acid sequence complementary to a) or b); and d) fragments of a),b), or c) that are at least 15 nucleotide bases in length and thathybridize under stringent conditions to DNA which encodes thepolypeptides set forth SEQ ID NO:
 2. 23. A method for detectingα1,2-fucosyltransferase polynucleotide in a sample, comprisingamplifying the α1,2-fucosyltransferase polynucleotide.
 24. The method ofclaim 23, wherein the polynucleotide is amplified using PCR.
 25. Arecombinant method for producing α1,2-fucosyltransferase polypeptide,comprising: inserting a nucleic acid comprising the polynucleotide ofclaim 7 adjacent to a selectable marker, such that the resultingpolynucleotide encodes a recombinant α1,2-fucosyltransferase polypeptidefused to the selectable marker.
 26. A polynucleotide produced by themethod of claim
 25. 27. A host cell containing the polynucleotide ofclaim
 25. 28. A recombinant method for producing α1,2-fucosyltransferasepolypeptide, comprising: a) culturing a recombinant host cell containinga polynucleotide encoding the α1,2-fucosyltransferase polypeptide underconditions which allow expression of α1,2-fucosyltransferasepolypeptide; and b) isolating the polypeptide.
 29. A method of producinga α1,2-fucosyltransferase fusion protein comprising: a) growing a hostcell containing a polynucleotide encoding α1,2-fucosyltransferasepolypeptide operably linked to a polynucleotide encoding a polypeptideor peptide of interest under conditions which allow expression of thefusion protein; and b) isolating the fusion protein.
 30. A geneexpression system for producing α1,2-fucosyltransferase comprising ahost cell modified with a polynucleotide encodingα1,2-fucosyltransferase polypeptide or an enzymatically active portionthereof.
 31. The gene expression system of claim 30, wherein thepolynucleotide is DNA.
 32. The gene expression system of claim 30,wherein the polynucleotide is cDNA.
 33. The gene expression system ofclaim 30, wherein the polynucleotide is RNA.
 34. The gene expressionsystem of claim 30, wherein the host cell is selected from the groupconsisting of a bacterial cell, a yeast cell, a fungal cell, a plantcell or an animal cell.
 35. The gene expression system of claim 30,wherein the host cell is recombinantly modified by transfection with aplasmid.
 36. The gene expression system of claim 35, wherein the plasmidcomprises a selectable marker.
 37. The gene expression system of claim36, wherein the selectable marker is glutamine synthetase.
 38. A methodfor producing α1,2-fucosyltransferase polypeptide, comprising the stepsof: (a) culturing a gene expression system comprising a host cellmodified with a polynucleotide encoding the α1,2-fucosyltransferasepolypeptide or an enzymatically active portion thereof; and (b)harvesting the α1,2-fucosyltransferase.
 39. The method of claim 38,further comprising substantially purifying the harvestedα1,2-fucosyltransferase polypeptide.
 40. The method of claim 38, whereinthe polynucleotide is DNA.
 41. The method of claim 38, wherein thepolynucleotide is cDNA.
 42. The method of claim 38, wherein thepolynucleotide is RNA.
 43. The method of claim 38, wherein the host cellis recombinantly modified by transfection with a plasmid.
 44. The methodof claim 43, wherein the plasmid comprises a selectable marker.
 45. Themethod of claim 44, wherein the selectable marker is glutaminesynthetase.
 46. The method of claim 38, wherein the host cell isselected from the group consisting of bacterial cell, yeast cell, fungalcell, plant cell or animal cell.
 47. A method for producing afucosylated oligosaccharide, the method comprising contacting aα1,2-fucosyltransferase polypeptide with an α1,2-fucosyltransferasesubstrate for a suitable time and under suitable conditions to producethe oligosaccharide.
 48. The method of claim 47, wherein the fucosylatedoligosaccharide is selected from the group consisting of Le^(B), Le^(y)or H type 1 and H type
 2. 49. The method of claim 47, wherein thesubstrate is LacNAc-R and GDP-fucose.
 50. The method of claim 47,wherein the oligosaccharide is purified.
 51. A method for producingfucosylated oligosaccharides, the method comprising the steps of: (a)culturing a gene expression system comprising a host cell modified witha polynucleotide encoding a α1,2-fucosyltransferase polypeptide or anenzymatically active portion thereof; and (b) contacting the host cellwith a substrate, under conditions and for sufficient time to producethe oligosaccharides.
 52. The method of claim 51, wherein thefucosylated oligosaccharide is selected from the group consisting ofLe^(B), Le^(y) or H type 1 and H type
 2. 53. The method of claim 51,wherein the substrate is LacNAc-R and GDP-fucose.
 54. The method ofclaim 51, wherein the oligosaccharide is purified.